Methods, immunoassays and devices for detection of anti-lipoidal antibodies

ABSTRACT

Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, a method for immobilizing a lipoidal antigen, comprising cardiolipin, lecithin, and cholesterol, on a solid support (such as a nitrocellulose membrane) is described. The ability to immobilize a lipoidal antigen on a membrane satisfies a long-felt need for membrane-based assay for the detection of anti-lipoidal antibodies. Also described are immunoassay devices for concurrently performing treponemal and non-treponemal tests for syphilis.

CROSS REFERENCE TO RELATED REFERENCES

This application claims the benefit of U.S. Provisional PatentApplication No. 60/693,120, filed on Jun. 21, 2005, which application ishereby incorporated by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made by the National Center for HIV, STD, and TBPrevention, Division of AIDS, STD, and TB Laboratory Research,Laboratory Reference and Research Branch, Centers for Disease Controland Prevention, an agency of the United States Government.

FIELD

This disclosure concerns methods of immobilizing a lipoidal antigen to asolid support and related immunoassays and immunoassay devices (such as,test strips, flow-through devices, or lateral flow devices), whichassays and devices are useful, for example, for detection ofanti-lipoidal antibodies and/or diagnosis of disease (such as,syphilis).

BACKGROUND

Syphilis is a sexually transmitted disease (STD) caused by thespirochete bacterium Treponema pallidum. Over 100,000 cases of adultsyphilis are reported worldwide each year. In addition, the disease istransmitted congenitally and affects 3,000 or more infants annually. Thecourse of syphilis infection spans many years and may lead to a varietyof clinical presentations, which are characterized by four stages.

The primary stage of syphilis infection occurs 10-100 days afterbacterial infection, and is characterized by the appearance of one ormore chancres (red, bloodless, painless ulcers typically less than 1 cmin diameter). The chancres may appear on the genitalia or elsewhere onthe body. A chancre lasts 3-6 weeks and heals without treatment, leavinga small scar. Infected persons are contagious during this stage.

The secondary stage of syphilis infection is characterized by rash-likeskin lesions that can cover part or all of the body. The skin lesionsare generally painless and appear 1-6 months after the onset of theinitial chancre(s). The skin lesions can resemble warts, pustules, orulcers. Left untreated, they heal in 2-12 weeks without scarring. Fever,sore throat, weakness, weight loss, swelling of the lymph nodes, andloss of the eyelashes and/or part of the eyebrows can also occur duringthis stage of infection. In addition, the symptoms may progress tomeningovascular syphilis, which is characterized by inflammation of thecovering of the brain and spinal cord and/or changes in the vasculatureof the brain. Infected persons are also contagious in the secondaryphase.

The next stage of this disease is latent syphilis or the hidden stage.During this stage, the infected person appears to have recovered and isasymptomatic. This stage lasts for life in approximately two-thirds ofpersons who are not treated for syphilis. During the first year oflatency, relapses of secondary stage symptoms may occur. Except during arelapse, infected persons are not contagious during this latent stage;however, children born to latently infected mothers within four years ofthe appearance of the primary chancre may contract congenital syphilis.

Tertiary or late syphilis is the final stage of untreated infection.This stage may occur as early as one year after infection or anytimethereafter with 10 to 20 years being most common. Benign syphilis,characterized by lesions called gummas, can occur in the bone, skin, andinternal organs. Death is rare, but severe disfigurement and pain canoccur. Cardiovascular syphilis is characterized by aortic aneurisms aswell as other cardiovascular problems and frequently results in death.Neurologic involvement may occur in the early stages of syphilis as wellas manifest as late stage symptoms. In the late stage disease,neurosyphilis may be asymptomatic or the patient may have severeneurologic problems such as possible dementia, insanity, impairment ofmobility, blindness, deafness, or even death.

The immune response in syphilis involves production of (i) treponemalantibodies, which are specific for T. pallidum antigens, and (ii)anti-lipoidal antibodies, which recognize lipoidal material releasedfrom damaged host cells, lipoprotein-like material and possiblycardiolipin released from the treponemes. The mainstay of syphilisscreening and diagnosis is serological testing for either or both ofthese two types of antibodies.

Tests for anti-lipoidal antibodies (often called “non-treponemal tests”)are typically based on an antigen composed of naturally occurringcardiolipin, cholesterol and lethicin. The widely used non-treponemaltests (e.g., Venereal Disease Research Laboratory (VDRL) test and RapidPlasma Reagin (RPR) test) monitor, either microscopically (e.g., VDRLtest) or macroscopically (e.g., RPR test), the formation of a flocculentcomprised of antigen-antibody complexes. Non-treponemal tests have theadvantage of being widely available, inexpensive and convenient toperform on large numbers of specimens. Moreover, because anti-lipoidalantibody titers decrease with successful treatment for syphilis,eventually disappearing in most patients, while treponemal antibodiestiters remain high for years or even a lifetime, non-treponemal testsare considered the better choice for monitoring treatment or testing forreinfection.

Treponemal tests are based on antigens derived from T. pallidum andinclude the T. pallidum particle agglutination (TP-PA), the fluorescenttreponemal antibody-absorbed test (FTA-ABS) and enzyme immunoassays.Treponemal tests are used primarily to verify reactivity innon-treponemal tests. The treponemal test may also be used to confirm aclinical impression of syphilis in which the non-treponemal test isnonreactive. Treponemal tests are technically more difficult, timeconsuming, and expensive to perform and cannot be used to monitortreatment because the test will remain reactive for years or a lifetimein approximately 85% of persons successfully treated for syphilis.

Each of the above-described antibody tests is performed using a serumsample that is obtained in a clinical setting and sent to a laboratoryfor analysis. Therefore, test results are typically not available forseveral days after the sample is collected. Because of the frequentdifficulty of tracing patients with STDs, the development of a rapid,point-of-care test is needed to aid the clinician in making a judgment,preferably on the day of testing.

Immunoassay devices (such as test strips, flow-through devices, orlateral flow devices), which offer rapid, on-site results, are availableto qualitatively test serum levels of treponemal antibodies (e.g.,DiaSys Corporation; ACON Laboratories, Inc.; Biokit, S.A.; GenixTechnology; Standard Diagnostics; Cortez Diagnostics, Inc.; and PhoenixBio-Tech Corp). However, analogous tests for anti-lipoidal antibodieshave been more difficult to develop at least in part because thehydrophobic antigens of the anti-lipoidal antibodies (e.g., VDRL, USR orRPR antigens, or cardiolipin) resist attachment to solid supports, whichis one element of an immunoassay device.

According to some experts, syphilis detection would be further aided bya combination of a non-treponemal test and a treponemal test forscreening and diagnostic purposes. This is an approach advocated by theWorld Health Organization, Treponemal Infections, Technical ReportSeries 674, Geneva: WHO, 1982. An easy-to-use, rapid, point-of-care testcapable of concurrently detecting both non-treponemal and treponemalantibodies would help address this long-felt need.

SUMMARY

Efforts to develop non-solution immunoassays for non-treponemal testing(or combined non-treponemal and treponemal testing) have been frustratedby the difficulty of attaching antigens specifically recognized byanti-lipoidal antibodies (referred to as “lipoidal antigens”), such ascardiolipin, VDRL antigen, USR antigen and the like, to a solidsubstrate, such as a nitrocellulose strip. For instance, the very smallsize of the cardiolipin molecule has resulted in poor localization ofthis molecule on a solid substrate. Although the size of the cardiolipinmolecule could be increased by conjugating cardiolipin to largermolecules (such as proteins), such conjugations have resulted in theloss of cardiolipin antigenicity. More generally, the high degree ofhydrophobicity of lipoidal antigens makes it difficult to bind suchantigens to many solid surfaces, such as nitrocellulose and othermicroporous membranes.

The present disclosure provides an approach for reliably attachinglipoidal antigens (which are made up of, at least, cardiolipin,phosphatidylcholine (also referred to as “lecithin”), and cholesterol)to a solid substrate (such as, a microporous membrane) while maintainingthe antigenicity and specificity of the antigen for anti-lipoidalantibodies. Using methods described herein, it is now possible to attachlipoidal antigen to a variety of solid supports. The ability to attachthe lipoidal antigen in this manner allows it to be used, for instance,in immunoassay devices for rapid, on-site testing of non-treponemalantibodies. In certain embodiments, disclosed immunoassay devices alsoincorporate T. pallidum antigens that are recognized by treponemalantibodies, such that the device conveniently and concurrently detectsboth non-treponemal (i.e., anti-lipoidal) and treponemal (i.e., anti-T.pallidum) antibodies.

In one particular example, a lipoidal antigen comprising cardiolipin,lecithin and cholesterol is contacted with a population of Fab fragmentsspecific for the lipoidal antigen to provide a lipoidal antigen-Fabcomplex, which complex is readily attachable to a solid support, such asa permeable substrate of a flow-through or lateral flow device.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tracing of the absorbance at 280 nm of the eluant from aProtein A column loaded with papain-digested purified IgG. An ISCO UA-5Absorbance/Fluorescence Detector set to sensitivity 2 and chart speed1.5 was used to collect the data. Glycine elution buffer was added tothe column when the absorbance at 280 nm of the eluant returned tobaseline after elution of Peak I.

FIG. 2 shows a digital image of an SDS gradient gel. Lanes (1) molecularweight standards (from top: 203, 135, 83, 41, 31, 17, and 7 kD); (2)ammonium sulfate precipitate from human syphilitic serum; (3) ammoniumsulfate precipitated proteins not retained by Protein A (peak I); (4)Protein A-purified IgG from ammonium sulfate precipitate (peak II); (5)Protein A-purified IgG before papain digestion; (6) Protein A-purifiedIgG after papain digestion; (7) proteins from papain IgG digestion thatwere not retained by Protein A (peak 1) (predominantly Fab fragments);(8) Protein A-retained proteins from papain IgG digestion (including Fcfragments); and (9) molecular weight standards (from top: 207, 116, 98,55, 37, 30, 20 and 7 kD).

FIG. 3 shows a series of nitrocellulose dipsticks prepared as describedin Example 7. The dipsticks were spotted with IgG or Fab or Fc fragmentsas indicated, and later dipped into a solution containinggold-conjugated Protein A.

FIG. 4 shows a series of nitrocellulose dipsticks prepared as describedin Example 8. The dipsticks were spotted with a USR antigen coated withthe indicated amount of Fab fragment, and later dipped into a solutioncontaining gold-conjugated Protein A and reactive syphilis serum (R) ornon-reactive syphilis serum (NR).

FIG. 5 is a graph of OD₅₈₀ for mixtures of rabbit anti-human IgG (Fc)with colloidal gold as a function of pH. As described in Example 9, auseful pH at which to obtain a stable gold conjugate for these reactantsis the pH corresponding to the lowest OD₅₈₀.

FIG. 6 is a graph of OD₅₈₀ for mixtures of rabbit anti-human IgG (Fc)with colloidal gold as a function of rabbit anti-human IgG (Fc)concentration. As described in Example 10, the protein concentrationproducing the lowest OD₅₈₀ represents a useful concentration of rabbitanti-human IgG (Fc) to associated with gold particles.

FIG. 7 shows a digital image of five different physical embodiments oflateral flow devices that could be used with the disclosed methods. Thedevice embodiments shown in (A), (B) and (E) are configured so that eachmay be dipped into, or partially submerged in, the sample or asample-containing solution. The device embodiments shown in (C) and (D)are configured so as to receive a volume of the sample (or asample-containing solution) dropwise into a sample introduction port.

FIG. 8 is a perspective view of a physical embodiment of a lateral flowdevice, with a portion of the housing broken away to show the basiccomponents of the device and their relationship to each other.

FIG. 9 is a schematic representation of one embodiment of an immobilizedlipoidal antigen and the capture of an anti-lipoidal antibody analyte.

FIG. 10 provides an illustration of an exemplary flow-through device forsimultaneous detection of treponemal and non-treponemal antibodies insyphilis. The device is configured to receive a volume of the sampledropwise into a sample introduction port (located in the center of thedevice).

DETAILED DESCRIPTION I. Introduction

Disclosed herein are immunoassay devices for determining the presenceand/or amount of an anti-lipoidal antibody in a fluid sample, such ashuman sera. Such devices include a microporous substrate (such asnitrocellulose, nylon, polyvinylidene fluoride (PVDF), polyethersulfone,polycarbonate, polyester, cellulose acetate, mixed cellulose esters, orcombinations thereof). The substrate includes, among other things, ananti-lipoidal capture area in which is immobilized a lipoidalantigen-anchor antibody complex. Such complex has an anchor antibodycomponent, which is immobilized on the substrate, and a lipoidal antigencomponent, which is specifically bound by the anchor antibody and isthereby anchored to the substrate. The lipoidal antigen componentincludes cardiolipin, lecithin and cholesterol and can be specificallybound by anti-lipoidal antibodies (such as reagin antibodies present inT. pallidum-infected subjects).

Other device embodiments further include a sample application area and aflow path from the sample application area to the anti-lipoidal capturearea. The sample application area is in fluid-continuous contact withthe anti-lipoidal capture area such that a fluid sample placed in thesample application area can flow through or along the membrane (forexample by capillary action or chromatographic flow) to theanti-lipoidal antibody capture area. The presence and/or amount of ananti-lipoidal antibody in the fluid sample can be detected by formationof a complex between the anti-lipoidal antibody and the immobilizedlipoidal antigen-anchor antibody complex. Some disclosed devices alsoinclude an absorbent pad, which is in contact with the membrane andserves as a reservoir for the sample after it contacts the capturearea(s). In particular examples, a disclosed device is a lateral flowdevice or a flow-through device.

In some embodiments, the anchor antibody is a Fab fragment specific forcardiolipin. In other cases, a plurality of Fab fragments produced fromimmunoglobulins isolated from one or more T. pallidum-infected subjectsserve as anchor antibodies. Some device embodiments include a lipoidalantigen that is a USR antigen, a VDRL antigen, or a synthetic VDRLantigen. In some cases (for example, for a flow-through device), asubstrate (such as a nitrocellulose membrane) has a pore size from about0.2 μm to about 8 μm. In other instances (for example, for alateral-flow device), a substrate (such as a nitrocellulose membrane)has a pore size up to about 12 μm. In still other device embodiments,the anti-lipoidal antibody capture area comprises one or more lines,which, in some examples, can have a width from about 8 mm to about 15mm.

In exemplary devices, the lipoidal antigen-anchor antibody complex isimmobilized on the membrane by a method involving (a) contacting thelipoidal antigen with one or more anchor antibodies specific for atleast one of cardiolipin, lecithin, or cholesterol to form the lipoidalantigen-anchor antibody complex; and (b) applying the lipoidalantigen-anchor antibody complex to the membrane. In more specificexamples, the lipoidal antigen-anchor antibody complex is immobilized onthe membrane by a method involving (i) immobilizing an anchor antibodyspecific for at least one of cardiolipin, lecithin, or cholesterol onthe membrane; (ii) blocking non-specific binding sites on the membrane;(iii) contacting the immobilized anchor antibody with lipoidal antigento form a lipoidal antigen-anchor antibody complex; and (iv) washing themembrane to remove any unbound lipoidal antigen.

Disclosed examples of the device provide an anchor antibody bound to anepitope naturally occurring in a component of the lipoidal antigen(e.g., cardiolipin or cholesterol) and not to a derivative groupintroduced into the antigen for the purposes of providing a binding sitefor the anchor antibody. For example, the anchor antibody does not bindto a biotinylated component of the lipoidal antigen (such asbiotinylated lecithin or cardiolipin).

In some embodiments, a disclosed immunoassay device further includes atreponemal capture area having either (a) an immobilized treponemalantigen capable of being specifically bound by an anti-T. pallidumantibody, or (b) an immobilized anti-T. pallidum antibody thatspecifically binds a mobile treponemal antigen. In these embodiments, afluid sample applied in the sample application area can flow through oralong the membrane to the anti-lipoidal antibody capture area and to thetreponemal capture area.

Also disclosed herein are lateral flow devices for determining thepresence and/or amount of an anti-lipoidal antibody in a fluid sample.These devices typically include a sample application area and a separateanti-lipoidal antibody capture area in which an immobilized anchorantibody-lipoidal antigen complex is provided which complex has aspecific binding affinity for a mobile-phase anti-lipoidal antibody. Anyliquid (such as a fluid biological sample) applied in the sampleapplication area flows along a path of flow from the sample applicationarea to the anti-lipoidal antibody capture area. The path of flow maycontinue to a downstream absorbent pad associated with the lateral flowdevice, which acts, at least in part, as a liquid reservoir. Formationof a complex between the anti-lipoidal antibody and the immobilizedlipoidal antigen complex can be detected to determine the presenceand/or amount of the anti-lipoidal antibody in a fluid sample.

In some embodiments of the lateral flow device, a conjugate pad isplaced in the path of flow from the sample application area to theanti-lipoidal antibody capture area. The conjugate pad includes a mobileor mobilizable detector reagent for an anti-lipoidal antibody, such thatflow of liquid through the pad moves the detector reagent to theanti-lipoidal antibody capture area. Formation of a complex among thedetector reagent, anti-lipoidal antibody, and the anchorantibody-lipoidal antigen complex provides a visible or otherwisedetectable indicator of the presence of the anti-lipoidal antibody in abiological specimen. In alternative embodiments, the detector reagent isnot supplied in a conjugate pad, but is instead applied to themicroporous membrane, for example from a developer bottle.

Examples of a detector reagent include a labeled Protein A, Protein G,or anti-human antibody, in which the label is one or more of an enzyme,colloidal gold particle, colored latex particle, protein-adsorbed silverparticle, protein-adsorbed iron particle, protein-adsorbed copperparticle, protein-adsorbed selenium particle, protein-adsorbed sulfurparticle, protein-adsorbed tellurium particle, protein-adsorbed carbonparticle, or protein-coupled dye sac.

Certain embodiments of the lateral flow device also include a treponemalcapture area in the flow path from the sample application area. Suchtreponemal capture area may include, for example, an immobilizedtreponemal antigen having a binding affinity for a mobile anti-T.pallidum antibody or an immobilized anti-T. pallidum antibody having abinding affinity for a mobile T. pallidum organism or T. pallidumantigen. The lateral flow device may also have a mobile or mobilizabledetector reagent specific for the treponemal antibody in the conjugatepad. The detector reagent for the treponemal antibody may be in the sameor a different pad than the detector reagent for the anti-lipoidalantibody. In particular embodiments, a detector reagent specific for ananti-T. pallidum antibody comprises gold-conjugated Protein A,gold-conjugated Fc-specific Protein G, or gold-conjugated anti-humanantibody (Fc portion). In other embodiments, a treponemal capture areacan include an anti-treponemal antibody for capture of a mobiletreponemal antigen present in a fluid sample. In such embodiments, adetector reagent for the mobile treponemal antigen can be a gold-labeledanti-treponemal antigen antibody.

The disclosed immunoassay devices (e.g., flow-through or lateral-flowdevice) can be used in methods for detecting anti-lipoidal antibodiesand/or diagnosing syphilis in a subject by applying a biological sample(such as, blood, serum, skin ulcer exudate, urine, saliva, or sputum)from a subject to a disclosed device and detecting formation of acomplex among the anti-lipoidal antibody, the anchor antibody-lipoidalantigen complex, and an anti-lipoidal antibody detector reagent in thecapture area. Detection of the formation of the complex in the capturearea detects an anti-lipoidal antibody associated with T. palliduminfection or the disease syphilis. In those embodiments in which thedevice includes a conjugate pad in the path of flow from the sampleapplication area to the cardiolipin capture area, the detected complexincludes the mobile or mobilizable detector. In other embodiments inwhich the detector reagent is applied to the device from an externalsource, the detected complex includes the externally applied detector.In some embodiments, a detector reagent is labeled Protein A,Fc-specific Protein G, or anti-human antibody.

In particularly advantageous embodiments of the method, the discloseddevice is capable of detecting both the anti-lipoidal antibodies (which,for example, are an indicator of active infection) and anti-treponemalantibodies (which, for example, verify reactivity of the non-treponemaltest). In those embodiments of the device which include the treponemalantigen, the method includes detecting formation of a complex betweenthe anti-T. pallidum antibody, the immobilized treponemal antigen, and adetector reagent in the capture area. As with the detector reagent usedfor anti-lipoidal antibodies, the detector reagent for the treponemalantibodies can be provided on the device or applied from an externalsource.

Also disclosed herein are methods for immobilizing immunoreactivecardiolipin on a solid support (such as, nitrocellulose). Such methodsinvolve (a) contacting a lipoidal antigen, comprising immunoreactivecardiolipin, with one or more antibodies specific for at least onecomponent of the lipoidal antigen to form a lipoidal antigen-antibodycomplex; and (b) applying the lipoidal antigen-antibody complex to asolid support; wherein applying the lipoidal antigen-antibody complex tothe solid support immobilizes the immunoreactive cardiolipin on thesolid support. In particular examples, the lipoidal antigen is a USRantigen, a VDRL antigen, or a synthetic VDRL antigen. In other examples,the antibody is an antibody fragment that will not substantially reactwith Protein A, Fc-specific Protein G, or anti-human antibody (Fcportion) (such as, a Fab fragment). In still other examples, theantibody is isolated from serum of a T. pallidum-infected or T.pallidum-inoculated subject.

Other exemplary methods for immobilizing immunoreactive cardiolipin on asolid support, involve (a) immobilizing one or more antibodies specificfor cardiolipin, lecithin and/or cholesterol on a solid support; (b)blocking non-specific binding sites on the solid support; (c) applying alipoidal antigen, comprising immunoreactive cardiolipin, lecithin and/orcholesterol, to the solid support to form lipoidal antigen-immobilizedantibody complexes; and (d) washing the solid support to remove anylipoidal antigen that is not specifically bound by the one or moreimmobilized antibodies.

Also disclosed herein are kits for the diagnosis of syphilis. These kitsinclude a disclosed device (such as a flow-through or lateral-flowdevice) and instructions for applying the biological sample to thesample application area or the device. The kit may also includeinstructions for interpreting results of the test.

The disclosed devices can be also used in methods for diagnosing lupusin a subject by analyzing a biological sample from the subject, byapplying the biological sample to the device and detecting formation ofa complex among the anti-lipoidal antibody, the anchor antibody-lipoidalantigen complex, and a detector reagent in the capture area. Detectionof the formation of the complex in the capture area detects ananti-lipoidal antibody associated with lupus. In some instances, one ormore co-factors (such as β₂-glycoprotein I) are present (such as, addedto a sample) for the detection of lupus.

II. Abbreviations and Terms

Ab antibody

HPLC high pressure liquid chromatography

LFD Lateral flow device

PEG polyethylene glycol

PVA polyvinyl alcohol

PVDF polyvinylidene fluoride

PVP polyvinyl pyrrolidone

SDS sodium dodecyl sulfate

USR unheated serum regain

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Analyte: A target, such as an atom, molecule, group of molecules orcompound of natural or synthetic origin (e.g., drug, hormone, enzyme,growth factor antigen, antibody, hapten, lectin, apoprotein, orcofactor) sought to be detected or measured that is capable of bindingspecifically to immobilized lipoidal antigens described herein. Analytesmay include, but are not limited to biological analytes, antibodies,drugs, hormones, antigens, haptens, lectins, apoproteins, or cofactors.In some embodiments, the analyte includes antibodies, such asanti-lipoidal antibodies (e.g., anti-cardiolipin antibodies), producedin response to infection by T. pallidum. In other embodiments, theanalyte includes anti-lipoidal antibodies produced in response to any of(i) an autoimmune disease, such as lupus, (ii) various venous andarterial thrombotic disorders, including cerebral infarction, (iii) deepvenous thrombosis, (iv) thrombocytopenia

Antibody: A protein consisting of one or more polypeptides substantiallyencoded by immunoglobulin genes or fragments of immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad of immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

Antibodies are naturally produced in plants and animals in response toantigens presented to the immune system. Naturally produced antibodiesmay be found, for example, in the serum of an animal. For example, aperson infected with T. pallidum typically will produce antibodies atleast against T. pallidum antigens and antibodies against lipoidalmaterial that results from the treponemal infection (e.g., anti-lipoidalantibodies), for example, from host cells damaged by the infection.

Antibodies may exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases or by recombinant DNA methods. Exemplary antibody fragmentsinclude, for example, Fab, Fab′, F(ab′)₂, Fv, Fd, dAb, complementaritydetermining regions (CDR), and single-chain antibodies (scFv). A Fabfragment is a monovalent fragment consisting of the VL, VH, CL and CH1domains; an F(ab′)₂ fragment is a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; an Fdfragment consists of the VH and CH1 domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragmentconsists of a VH domain (see, e.g., Ward et al., Nature, 341: 544-546,1989; Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y., 1993).

While certain antibody fragments are defined in terms of the digestionof an intact antibody, it will be appreciated that Fab fragments orother antibody fragments may be synthesized de novo either chemically orby utilizing recombinant DNA methodology. Thus, the term antibody asused herein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies.

Antigen: A chemical or biochemical compound, composition, structure,determinant, antigen or portion thereof that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

[Antigen-specific] Hyperimmune serum: Polyclonal antiserum havingspecificity for a particular antigen (such as, a lipoidal antigen and/orcardiolipin), which antiserum is produced in a subject in response torepeated challenge (e.g., by injection, infection or other route) withthe antigen of interest (or an organism or other composition containingor producing the antigen of interest).

Anti-lipoidal antibody: An antibody (such as IgM or IgG) havingspecificity for a lipoidal antigen (see below). In some instances,anti-lipoidal antibodies are produced by the immune system of a subject(such as a human) in response to a disease state, such as a microbial(e.g., bacterial) infection. For example, this term contemplatesanti-lipoidal antibodies produced as a consequence of T. palliduminfection. Although not bound by theory, it is thought thatanti-lipoidal antibodies in subjects infected with T. pallidum areproduced as the result of lipids released from host cells and/orlipoprotein-like material and possibly cardiolipin released fromtreponemes. Anti-lipoidal antibodies may also be produced in response tonon-treponemal diseases, including, for example (i) an autoimmunedisease, such as lupus (Harris et al., Clin. Rheum. Dis., 11:591-609,1985), (ii) various venous and arterial thrombotic disorders, includingcerebral infarction (Harris et al., Clin. Exp. Rheumatol., 2:47-51,1984), (iii) deep venous thrombosis (Mueh et al., Ann. Intern. Med.,92:156-159, 1980), (iv) thrombocytopenia (Harris et al., Clin. Rheum.Dis., 11:591-609, 1985), (v) pulmonary embolism (Anderson and Ali, Ann.Rheum. Dis., 43:760-763, 1984), or (vi) recurrent fetal loss withplacental infarction (Derue et al., J. Obstet. Gynaecol., 5:207-209,1985). Anti-lipoidal antibodies found in non-treponemal diseases mayspecifically bind a lipoidal antigen in the presence of one or moreco-factors (such as β₂-glycoprotein I).

Anti-lipoidal antibodies for use in some embodiments of the methods andcompositions (e.g., devices) disclosed herein can be of any derivation,but often will be found in the serum of a subject.

Binding affinity: A term that refers to the strength of binding of onemolecule to another at a site on the molecule. If a particular moleculewill bind to or specifically associate with another particular molecule,these two molecules are said to exhibit binding affinity for each other.Binding affinity is related to the association constant and dissociationconstant for a pair of molecules, but it is not critical to theinvention that these constants be measured or determined. Rather,affinities as used herein to describe interactions between molecules ofthe described methods and devices are generally apparent affinities(unless otherwise specified) observed in empirical studies, which can beused to compare the relative strength with which one molecule (e.g., anantibody or other specific binding partner) will bind two othermolecules (e.g., an analyte and an analyte-tracer conjugate). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

Biological Sample: Any sample that may be obtained directly orindirectly from a subject, including whole blood, plasma, serum, tears,mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat,semen, vaginal secretion, sputum, fluid from ulcers and/or other surfaceeruptions (such as blisters, or abscesses), amniotic fluid, synovialfluid, cerebrospinal fluid, and/or extracts of tissues, cells or organs.The biological sample may also be a laboratory research sample such as acell culture supernatant. The sample is collected or obtained usingmethods well known to those skilled in the art.

Capture area: A region of an immunoassay device (such as a flow-throughdevice or lateral flow device) where a capture reagent (such as ananchor antibody-lipoidal antigen complex) is immobilized. An immunoassaydevice may have more than one capture area, for example, a “primarycapture area,” a “secondary capture area,” and so on. Often a differentcapture reagent will be immobilized in the primary, secondary, or othercapture areas. Multiple capture areas may have any orientation withrespect to each other on the substrate; for example, a primary capturearea may be distal or proximal to a secondary (or other) capture areaand visa versa. Alternatively, a primary capture area and a secondary(or other) capture area may be oriented perpendicularly to each othersuch that the two (or more) capture areas form a cross or a plus sign orother symbol.

Anti-lipoidal Antibody Capture Area: A capture area wherein an antigencapable of being specifically bound by an anti-lipoidal antibody (suchas a lipoidal antigen-anchor antibody complex) is immobilized as thecapture reagent.

Conjugate: When used in the verb form, the term “conjugate” means thecoupling of one molecule (e.g., Protein A, Protein G, or anti-human IgG(Fc) antibody) to another molecule (e.g., colloidal gold). Such couplingmay involve covalent or non-covalent (such as electrostatic or other)interactions between the components of the conjugate. Such coupling maybe achieved by chemical means, either with or without the use of alinking group. When used in the noun form, the term “conjugate” means acoupled molecular complex formed by conjugation.

Detecting or Detection: Refers to quantitatively or quantitativelydetermining the presence of the analyte(s) under investigation (e.g.,anti-lipoidal antibodies and/or anti-T. pallidum antibodies ortreponemal antigens). “Detecting Formation of a Complex” refers todetecting a complex comprising a detector reagent by any method suitablefor observing the particular label associated with the detector reagent;for instance, visual observation of a colored (or otherwise visible)label, measurement or visual detection of a fluorescent,chemiluminescent or radioactive label.

Detector Reagent (or Detection Agent): A reagent (or series of reagents)that permits the specific detection of a complex between an analyte(such as an anti-lipoidal antibody) and a capture reagent (such asanchor antibody-lipoidal antigen complexes). Further description ofdetector reagents and specific exemplary detector reagents are providedbelow.

Emulsion: A mixture of two immiscible fluids in which one phase ispresent as a colloidal dispersion in the other phase, which is referredto as the continuous, dispersing or solvent phase. Some emulsions willreadily separate when they are allowed to stand undisturbed. Otheremulsions may remain mixed for considerable lengths of time.

Epitope (or antigenic determinant): A site on the surface of an antigenmolecule to which an antibody molecule binds; generally an antigen hasseveral or many different antigenic determinants and reacts withantibodies of many different specificities. An “exogenous epitope” is anepitope that is not naturally found in an antigen molecule of interestand which is typically added to the antigen molecule to serve as abinding site for an antibody specific for (or other specific bindingpartner of) the exogenous epitope. An antigen molecule can be chemicallyor otherwise modified to include an exogenous epitope. For example, anantigen molecule can be biotinylated (i.e., derivatized with biotin)thereby producing an exogenous biotin epitope that can be specificallybound by an anti-biotin antibody or other biotin-specific bindingpartner (such as, avidin or strepavidin). In some instances, anexogenous epitope is an “epitope tag.” Epitope tags include peptidesequences (typically less than about 15 amino acids, but can be evenfull-length protein sequences) to which an antibody can specificallybind. Commonly known epitope tags include hexa-His, octa-His, FLAG, HA,and numerous others.

Immunogenicity: The property of being able to evoke an immune responsewithin an organism. For example, cardiolipin retains immunogenicity whenan anti-lipoidal antibody has the ability to bind an epitope present inthe cardiolipin.

Label: Any molecule or composition bound to an analyte, analyte analog,detector reagent, or binding partner that is detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Examples of labels, including enzymes,colloidal gold particles, colored latex particles, have been disclosed(U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and 4,954,452, eachincorporated by reference herein). Additional examples of useful labelsinclude, without limitation, radioactive isotopes, co-factors, ligands,chemiluminescent or fluorescent agents, protein-adsorbed silverparticles, protein-adsorbed iron particles, protein-adsorbed copperparticles, protein-adsorbed selenium particles, protein-adsorbed sulphurparticles, protein-adsorbed tellurium particles, protein-adsorbed carbonparticles, and protein-coupled dye sacs. The attachment of a compound(e.g., a detector reagent) to a label can be through covalent bonds,adsorption processes, hydrophobic and/or electrostatic bonds, as inchelates and the like, or combinations of these bonds and interactionsand/or may involve a linking group.

Lipoidal Antigen: An antigen including (but not limited to) cardiolipin,lecithin and cholesterol that is capable of being specifically bound byanti-lipoidal antibodies. The nature of cardiolipin, lecithin andcholesterol as contemplated by the term “lipoidal antigen” is discussedin detail elsewhere in the specification.

Microporous membrane: A thin film or structure having microscopic-sizedpores. Microporous membranes at least permit fluids and soluble analytesto travel along or through the membrane, for example by capillaryaction. Membranes are made of a wide variety of materials or compositesof materials, including, for example, polyethersulfone, nylon,polycarbonate, polyester, cellulose acetate, mixed cellulose esters,polyvinylidene fluoride, and/or nitrocellulose. Non-limiting suitablepore sizes can be up to about 0.22 micron, from about 0.22 micron toabout 0.45 micron, from about 0.22 micron to about 0.65 micron, fromabout 0.22 micro to about 0.8 micron, from about 0.22 micron to about1.2 micron, from about 0.65 micron to about 3 micron, from about 0.65micron to about 5 micron, from about 0.65 micron to about 8 micron, fromabout 5 micron to about 10 micron, or from about 5 micron to about 20micron. In particular instances, pore size can be about 0.22 micron,about 0.45 micron, about 0.65 micron, about 0.8 micron, about 1.2micron, about 3 micron, about 5 micron, about 8 micron, about 10 micron,or about 20 micron. Any membrane known to those of ordinary skill in theart to be suitable for lateral-flow or flow-through devices isenvisioned to be within the scope of the term “microporous membrane.”

Protein A and Protein G: Protein A is a protein isolated fromStaphylococcus aureus. Protein G is a protein isolated from aStreptococcus species. Both proteins have binding sites for the Fcportion of mammalian IgG. Native Protein G also contains binding sitesfor albumin, the Fab region of Igs, and membrane binding regions, whichcan lead to nonspecific binding; however, recombinant Protein G has beenengineered to eliminate the albumin, Fab, and membrane binding siteswhile retaining the Fc binding site, which makes it specific for the Fcportion of IgG. As used herein “Protein G” refers to the Fc-specificrecombinant form of Protein G (also referred to as “Fc-specific ProteinG”).

Specific binding partner (or binding partner): A molecule or compositioncapable of recognizing and binding to a specific structural aspect ofanother molecule or composition. Typical pairs of specific bindingpartners include antigen/antibody, hapten/antibody, hormone/receptor,nucleic acid strand/complementary nucleic acid strand, substrate/enzyme,inhibitor/enzyme, carbohydrate/lectin, biotin/(strept)avidin, andvirus/cellular receptor.

The phrase “specifically binds to” refers to the ability of a firstmolecular species (such as an antibody) to preferentially bind to asecond molecular species (such as an antigen recognized by the antibody)in comparison to other non-specific molecular species. Accordingly, thephrase “capable of being specifically bound by” refers to the ability ofa first molecular species (such as an antigen) to be preferentiallyrecognized and bound by a second molecular species (such as an antibodyspecific for the antigen) in comparison to other non-specific molecularspecies.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means “including A or B,” or “including A and B.” It is furtherto be understood that all molecular weight or molecular mass values areapproximate and are provided solely for description. Suitable methodsand materials used in the practice or testing of the disclosed subjectmatter are described below; however such materials and methods areillustrative only and not intended to be limiting. Methods and materialssimilar or equivalent to those described herein also can be used. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety to theextent permitted by applicable rules. In case of conflict, the presentspecification, including explanations of terms, will control.

III. Lipoidal Antigen

A lipoidal antigen is any antigen containing (without limitation)cardiolipin, lecithin and cholesterol, which antigen is capable ofspecifically binding an anti-lipoidal antibody. In some examples, alipoidal antigen specifically binds an anti-lipoidal antibody (e.g., ananti-cardiolipin antibody) present in a T. pallidum-infected subject(such as, a human). Lipoidal antigens comprising cardiolipin (CL),lecithin (L) and cholesterol (Ch) have long been used in solution assaysto detect anti-lipoidal antibodies in serum (see, e.g., prior discussionof “non-treponemal tests” for syphilis diagnosis). Specific exemplaryCL/L/Ch-containing lipoidal antigens previously used in solution assaysinclude the commonly known VDRL, RPR, and USR antigens (e.g., VenerealDisease Research Laboratory (VDRL) Slide Test, In: Larsen et al. (ed.),A Manual of Tests for Syphilis, 9th Edition, Washington D.C.: AmericanPublic Health Association, 1998, pp. 157-178; Pettit et al., “Unheatedserum reagin [USR] test as a quantitative test for syphilis,” J. Clin.Microbiol., 15(2): 238-242, 1982), and a synthetic form of the VDRLantigen (“Synthetic VDRL”) described by Castro et al. (Clin. Diagn. Lab.Immunol., 7(4):658-661, 2000). Unfortunately, the lipoidal nature ofsuch antigens has made it difficult to reliably attach any of theseantigens to a solid support, such as a bibulous (e.g., microporous)membrane, like nitrocellulose or others. Accordingly, there has been along-felt (but previously unmet) need to provide membrane-based assays(such as, flow-through or lateral flow immunoassay devices) fordetection of anti-lipoidal antibodies, for instance, in biologicalsamples. Such immunoassay devices would revolutionize, for example,point-of-care diagnosis of syphilis and other diseases characterized bythe presence of anti-lipoidal antibodies. This disclosure describes,among other things, a method for immobilizing lipoidal antigens, such asthe VDRL, USR, or Synthetic VDRL antigens, to membranous supports; thus,providing a means to satisfy a long-felt need in the art.

As discussed above, methods and compositions described hereincontemplate a lipoidal antigen that includes (e.g., comprises orconsists essentially of) cardiolipin, cholesterol and lecithin. Theseexemplary components of a lipoidal antigen are described in more detailbelow.

A. Cardiolipin

Cardiolipin is diphosphatidyl glycerol (specifically,1,3-diphosphatidylglycerol), which has a backbone consisting of threemolecules of glycerol joined by two phosphodiester bridges. The fourhydroxyl groups of cardiolipin's external glycerol moieties are eachesterified with a saturated or unsaturated fatty acid chain (typicallyfrom 14 to 18 carbons in length). As used herein, the term “cardiolipin”contemplates 1,3-diphosphatidylglycerol having any distribution of fattyacid side chains. Thus, the four fatty acid side chains of cardiolipincan independently vary in length (e.g., from about 14 to about 25carbons, from about 14 to about 22 carbons, from about 14 to about 20carbons, from about 14 to about 18 carbons, or from about 14 to about 16carbons) and/or degree of saturation (e.g., from completely saturated tohaving about 6 double bonds, from completely saturated to having about 4double bonds, or from completely saturated to having about 2 doublebonds). Exemplary fatty acid side chains of cardiolipin, as contemplatedherein, independently include myristoyl (14:0); palmitoyl (16:0);stearoyl (18:0); oleoyl (18:1); myristoleoyl (14:1); palmitoleoyl(16:1); petroselinoyl (18:1); linoleoyl (18:2); linolenoyl (18:3);eicosenoyl (20:1); arachidonoyl (20:4); erucoyl (22:1); DHA (22:6); ornervonoyl (24:1).

In some embodiments, cardiolipin is a naturally occurring form ofcardiolipin. The fatty acid composition of naturally occurringcardiolipin is generally distributed according to a wide variety ofnatural occurring fatty acids such as palmitoyl (16:0); stearoyl (18:0);oleoyl (18:1); and linoleoyl (18:2). The most abundant fatty acidmolecular species in naturally occurring forms of cardiolipin arelinoleic acid at 90%, followed by oleic acid at 5%, and palmitric acidat 1%. In other embodiments, cardiolipin is a non-naturally occurringform (also referred to as “synthetic cardiolipin”). Non-limitingexamples of synthetic cardiolipin include, for example, tetramyristoylcardiolipin, tetraoleoyl cardiolipin, tetramyristoyl-bis-(L-α-glyceryl)phosphoric acid, his (dipalmitoyl D,L-α-glycerylphosphoryl)-1,3 glycerolbenzyl ether disodium salt, his (dipalmitoylD,L-α-glycerylphosphoryl)-1,5 pentanediol disodium salt, his(dipalmitoyl D,L-α-glycerylphosphoryl)-1,3 propanediol disodium sal,bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,4 butanediol disodium salt,bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,2 ethanediol disodium salt,his (dipalmitoyl D,L-α-glycerylphosphoryl)-methanediol disodium salt,bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,3 glycerol disodium salt,his (benzylphosphoryl)-1,3-propanediol disodium salt, orD,L-α-dipalmitoyl bis-phosphatidic acid.

“Immunoreactive cardiolipin” is any form of cardiolipin (e.g., naturallyoccurring or synthetic cardiolipin) that specifically reacts withanti-lipoidal antibodies, such as anti-lipoidal antibodies present in asubject having syphilis or infected with T. pallidum.

B. Lethicin

Lecithin is the common name for phosphatidylcholine. Phosphatidylcholineis a glycerophospholipid, which is usually the most abundantphospholipid in animal and plants. It is a key building block ofmembrane bilayers, and is also the principal phospholipid circulating inthe plasma. Phosphatidylcholine contains two fatty acid side chains. Asused herein, the term “lecithin” contemplates phosphatidylcholine havingany distribution of fatty acid side chains. Thus, the two fatty acidside chains of cardiolipin can independently vary in length (e.g., fromabout 14 to about 25 carbons, from about 14 to about 22 carbons, fromabout 14 to about 20 carbons, from about 14 to about 18 carbons, or fromabout 14 to about 16 carbons) and/or degree of saturation (e.g., fromcompletely saturated to having about 6 double bonds, from completelysaturated to having about 4 double bonds, or from completely saturatedto having about 2 double bonds). Exemplary fatty acid side chains oflecithin, as contemplated herein, independently include myristoyl(14:0); palmitoyl (16:0); stearoyl (18:0); oleoyl (18:1); myristoleoyl(14:1); palmitoleoyl (16:1); petroselinoyl (18:1); linoleoyl (18:2);linolenoyl (18:3); eicosenoyl (20:1); arachidonoyl (20:4); erucoyl(22:1); DHA (22:6); or nervonoyl (24:1).

In some embodiments, lecithin is a naturally occurring form of lecithin.The fatty acid composition of naturally occurring lecithin includespalmitoyl (16:0), palmitoleoyl (16:1), linoleoyl (18:2), stearoyl(18:0), arachidonoyl (20:4), myristoyl (14:0), oleoyl (18:1), andlinolenoyl (18:3). The relative percentages of fatty acids in naturallyoccurring lecithin vary depending upon the source of the lecithin (e.g.,Balint et al., J. Lipid Res., 6(1):96, 1965). For example, lecithin inhuman gallbladder bile is reported to be about 45% 16:0, 4% 16:1, 4%18:0, 16% 18:1, 23% 18:2, 4% 20:4 with traces of 18:3 and 14:0; andlecithin in human plasma is reported to be about 35% 16:0, 1% 16:1, 14%18:0, 17% 18:1, 17% 18:2, 14% 20:4 with traces of 18:3 and 14:0. Inother embodiments, lecithin is a non-naturally occurring form (alsoreferred to as “synthetic lecithin”).

Non-limiting examples of lecithin include, for example:

Carbon Number 1-Acyl 2-Acyl 14:0-14:0 Myristoyl Myristoyl 14:0-16:0Myristoyl Palmitoyl 14:0-18:0 Myristoyl Stearoyl 16:0-14:0 PalmitoylMyristoyl 16:0-16:0 Palmitoyl Palmitoyl 16:0-18:0 Palmitoyl Stearoyl16:0-18:1 Palmitoyl Oleoyl 16:0-18:2 Palmitoyl Linoleoyl 16:0-20:4Palmitoyl Arachidonoyl 16:0-22:6 Palmitoyl Docosahexaenoyl 18:0-14:0Stearoyl Myristoyl 18:0-16:0 Stearoyl Palmitoyl 18:0-18:0 StearoylStearoyl 18:0-18:1 Stearoyl Oleoyl 18:0-18:2 Stearoyl Linoleoyl18:0-20:4 Stearoyl Arachidonoyl 18:0-22:6 Stearoyl Docosahexaenoyl18:1-14:0 Oleoyl Myristoyl 18:1-16:0 Oleoyl Palmitoyl 18:1-18:0 OleoylStearoyl 14:1-14:1 Myristoleoyl Myristoleoyl 14:1-14:1 MyristelaidoylMyristelaidoyl 16:1-16:1 Palmitoleoyl Palmitoleoyl 16:1-16:1Palmitelaidoyl Palmitelaidoyl 18:1-18:1 Petroselinoyl Petroselinoyl18:1-18:1 Oleoyl Oleoyl 18:1-18:1 Elaidoyl Elaidoyl 18:2-18:2 LinoleoylLinoleoyl 18:3-18:3 Linolenoyl Linolenoyl 20:1-20:1 EicosenoylEicosenoyl 20:4-20:4 Arachidonoyl Arachidonoyl 22:1-22:1 Erucoyl Erucoyl22:6-22:6 DHA DHA 24:1-24:1 Nervonoyl Nervonoyl

Other particular lecithin examples include, without limitation,DL-α-dimyristoyl lecithin, L-α-dimyristoyl cephalin, L-α-dipalmitoyllecithin, dipalmitoyl L-α-glycerophosphoric acid monocholine salt,L-α-dimyristoyl lecithin, D-α-dimyristoyl lecithin, L-α-distearoyllecithin, stearoyl glycollecithin,1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1),1,2-dilino1eoyl-sn-glycero-3-phosphocholine (18:2),1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0),1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15:0),1,2-diphytanoyl-sn-glycero-3-phosphocholine (16:0),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0), and1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

C. Cholesterol

Cholesterol is a steroid having, in one embodiment, the followingstructure:

Cholesterol is known to interact with phospholipids (such as,cardiolipin and lecithin) in membranes or membrane-like structures (suchas liposomes or micelles). In these circumstances, the cholesterolmolecule is believed to be oriented parallel to the fatty acid chains ofthe phospholipids, and the cholesterol hydroxyl group interacts with thephospholipid head groups.

In the formation of the lipoidal antigens disclosed herein, cholesterolis believed to stabilize the emulsion (e.g, micelle). Thus, for thepurposes of this disclosure, the term “cholesterol” includes any form ofcholesterol or other cholesterol derivative that is capable ofstabilizing an emulsion which contains a lipoidal antigen. In someexamples, stabilizing a lipoidal-antigen-containing emulsion refers toextending the time an emulsified lipoidal antigen remains in solution.For example, addition of cholesterol to a lipoidal antigen may extendthe time such an antigen remains dispersed in a continuous phase of anemulsion (such as, in an ethanolic solution) by 5%, 10%, 15% or 25% ascompared to a comparable antigen containing relatively less or nocholesterol.

Non-limiting examples of cholesterol derivatives include cholest-5-en-3beta-yl-6-aminohexyl ether (AH-Chol; Zimmer et al., Eur. J. Pharm.Biopharm., 47(2):175-8, 1999); poly(ethylene glycol) cholesteryl ethers(PEG(n)-Chols; Baba et al., Traffic, 2(7):501-12, 2001; Ishiwata et al.,Biochim. Biophys. Acta, 1359(2):123-35, 1997); a cationic cholesterolderivative with a hydroxyethyl amino head group described by Nakanishiand Noguchi (Adv. Drug Deliv. Rev., 52(3):197-207, 2001); cholesterolhemisuccinate (Meuillet et al., Eur. J. Pharmacol., 377(2-3):241-52,1999; dehydroergosterol (DHE), which differs from cholesterol in havingthree additional double bonds and an extra methyl group (Mukherjee,Biophys. J., 75(4):1915-1925, 1998); cationic derivatives of cholesterolwhich contain a tertiary amino head group with a different spacer arm(Takeuchi et al., FEBS Lett., 397(2-3):207-9, 1996); cholesteryl-3beta-carboxyamidoethylenedimethylamine (Noguchi et al., FEBS Lett.,433(1-2):169-173, 1998); and N-[tris[(beta-D-galactopyranosyloxy)methyl]methyl]-N alpha-[4-(5-cholesten-3beta-yloxy)succinyl]glycinamide (Kempen et al., J. Medicin. Chem.,27:1306-1312, 1984). Further examples of cholesterol derivatives usefulin the formation of membrane and membrane-like (e.g., liposomal ormicellar) structures (like a disclosed lipoidal antigen) are describedin U.S. Pat. Nos. 5,888,821; 5,043,164; 4,900,549; 4,442,037; 4,157,391;and 4,544,545, and European Pat. No. EP0606613.

D. Preparation of Lipoidal Antigen

In CL/L/Ch lipoidal antigen embodiments, cardiolipin, lecithin andcholesterol can be combined in any proportion that forms an antigencapable of being specifically bound by an anti-lipoidal antibody (suchas, anti-cardiolipin antibody). In some instances, the lipoidal antigenwill take the form of a micelle, liposome, membrane raft, or othermembrane-like structure. In these structures, hydrophobic interactionscause the non-polar components (such as, the fatty acid chains) toaggregate and exclude water molecules from the “core.” As one ofordinary skill in the art will appreciate, a micelle is a substantiallyspherical (or otherwise closed) non-bilayer structure having ahydrophobic interior composed of fatty acid chains (and not including anaqueous center). In comparison, a liposome is a substantiallyspherically arranged bilayer structure that is larger than a micelle andencloses an aqueous center. The type of structure formed by a CL/L/Chmixture will depend, for example, on the length and degree of saturationof the fatty acid chains of cardiolipin and lecithin, on thetemperature, on the ionic composition of the aqueous medium, and on themode of dispersal of the phospholipids in the solution. In particularembodiments, a CL/L/Ch lipoidal antigen (such as, a VDRL, RPR, USR, orsynthetic VDRL antigen) forms a micelle in an aqueous solution (such asan ethanolic solution). In more particular embodiments, a lipoidalantigen forms a micelle and does not form a liposome in an aqueoussolution (such as an ethanolic solution).

A lipoidal antigen for use in a disclosed composition or method can becommercially obtained (e.g., Fisher (Cat. No. B40765), Fisher (Cat. No.22-415-132), Fisher (Cat. No. 23-038010), True-Medix, IPX OverseasCorporation (Miami, Fla., USA), Cenogenics Corporation (Morganville,N.J., USA), Nova Century Scientific (Niagara Falls, N.Y., USA) as wellas other suppliers) or produced by any method commonly known in the art.In one embodiment, a lipoidal antigen is produced as described inExample 1.

In some embodiments, a non-aqueous solution containing cardiolipin,lecithin and cholesterol (CL/L/Ch) is mixed with an aqueous solution toform an emulsion. Any non-aqueous solution in which cardiolipin,lecithin and cholesterol are each soluble (or partially soluble) can beused, including, for example, ethanol (such as absolute ethanol, 95%ethanol, or 70% ethanol), chloroform, hexane:ethanol (e.g., 9:1),toluene (e.g., 95%), dichloromethane, or benzene. In one example, aCL/L/Ch is prepared in absolute ethanol.

In some embodiments, a CL/L/Ch solution useful for preparing a lipoidalantigen can include from about 0.001% to about 0.1% cardiolipin (such asfrom about 0.005% to about 0.07%, from about 0.008% to about 0.05%, fromabout 0.01% to about 0.04%, or from about 0.025% to about 0.035%); fromabout 0.05% to about 0.5% lecithin (such as from about 0.07% to about0.4%, from about 0.09% to about 0.3%, from about 0.1% to about 0.25%, orfrom about 0.14% to about 0.21%); and/or from about 0.2% to about 5%cholesterol (such as from about 0.4% to about 3%, from about 0.7% toabout 2%, from about 0.8% to about 1%). In particular examples, aCL/L/Ch solution useful for preparing a lipoidal antigen includes about0.03% cardiolipin (such as, naturally occurring cardiolipin), about0.21% lecithin (such as, naturally occurring lecithin), and about 0.9%cholesterol. In another example, a CL/L/Ch solution useful for preparinga lipoidal antigen includes about 0.03% tetramyristoyl cardiolipin,about 0.14% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and about0.9% cholesterol.

A CL/L/Ch solution useful for preparing a lipoidal antigen is mixed withan aqueous solution to form an emulsion of lipoidal antigen. The aqueoussolution can include any solution in which a CL/L/Ch solution isimmiscible, such as a saline solution (e.g., including 0.1-1.0 M NaCl,such as about 0.55 M NaCl). One exemplary aqueous solution includes 0.55M NaCl, 0.05% (v/v) formaldehyde, 0.26 mM, 0.35 mM to 0.66 mM disodiumhydrogen phosphate (for the dodecahydrate (12-H₂O), heptahydrate(7-H₂O), or anhydrous forms, respectively), and 1.2 mM potassium.

The ratio of CL/L/Ch solution to aqueous solution can be any ratio thatwill result in the formation of a lipoidal antigen emulsion (such as,CL/L/Ch-containing micelles). In some examples, the ratio of CL/L/Chsolution to aqueous solution ratio is about 1:10, about 1:15, about2:33, or about 1:20. In other examples, the percentage of CL/L/Chsolution in an emulsion is about 3% (v/v), about 5% (v/v), about 8%(v/v), about 10% (v/v), or about 12% (v/v).

IV. Methods of Immobilizing Lipoidal Antigen

It is known that anti-lipoidal antibodies (e.g., reagin) in human serumwill react with cardiolipin, lecithin and cholesterol-containing lipidantigens (such as, the RPR, VDRL, USR, or synthetic VDRL antigens). Asdiscussed above, this knowledge formed the basis for solution-based,non-treponemal serological tests. Despite a long-felt need formembrane-based assays for syphilis testing, no one had previouslyrecognized that a CL/L/Ch-containing lipoidal antigen could beimmobilized on a membrane (or other solid support) using, as oneexample, the anti-lipoidal antibodies the antigen was specificallydesigned to detect. Unlike many other antigens, which have only onebinding site for the antibody to be detected, the lipoidal antigen hasmany binding sites for anti-lipoidal antibodies. Thus, as disclosedherein, a non-saturating amount of anti-lipoidal antibody or otherantibody that can specifically bind a lipoidal antigen (or, inparticular examples, fragments of such antibodies, such as Fabfragments) can be used to anchor a lipoidal antigen to a solid support,such as a membranous surface (including, nitrocellulose, nylon andothers) without adversely affecting the ability of the immobilizedlipoidal antigen to further specifically bind anti-lipoidal antibodiespresent in a mobile phase (such as a biological sample).

A. Anchor Antibody

Antibodies useful to immobilize a lipoidal antigen to a solid support(such as a microporous membrane) in the disclosed compositions andmethods include any antibody that is capable of specifically binding alipoidal antigen, including, for example, anti-lipoidal antibodies froma T. pallidum-infected or T. pallidum-inoculated subject (such as, ahuman or a rabbit), anti-cholesterol antibodies, anti-cardiolipinantibodies, or anti-lecithin antibodies. As discussed in more detailbelow, antigen-binding fragments (such as, Fab fragments) of antibodieshaving the foregoing specificities can also serve as an anchor antibodyin the disclosed compositions and methods. Anchor antibodies may bemonoclonal or polyclonal. In specific embodiments, anchor antibodies arepolyclonal (such as those antibodies isolated from hyperimmune serumfrom T. pallidum-infected humans or rabbits).

Polyclonal anti-lipoidal antibodies from T. pallidum-infected or T.pallidum-inoculated subjects can be obtained, for example, by isolatingsuch antibodies from commercially available serum or by producing andisolating such polyclonal antibodies using methods commonly known in theart. Commercial suppliers of blood products (such as, serum) from T.pallidum-infected humans include New York Blood Center (New York, N.Y.,USA); Biomedical Resources (Hatboro, Pa., USA); Life Diagnostics, adivision of Life Therapeutics (formerly Serologicals) (Clarkston, Ga.,USA); and Teragenix (formerly Millennium Biotech, Inc) (Ft. Lauderdale,Fla., USA). In other embodiments, polyclonal antisera containinganti-lipoidal antibodies can be produced by immunizing host animals(such as rabbits, mice, horses, goats and others) with T. pallidumand/or a lipoidal antigen (such as a VDRL antigen). Detailed proceduresfor monoclonal or polyclonal antibody production are described in Harlowand Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).Specific non-limiting procedures for producing antibodies in a hostanimal using a CL/L/Ch-containing immunogen have been described by,e.g., Inoue and Nojima (Biochim. Biophys. Acta, 144(2):409-414, 1967).Specific non-limiting protocols are available for producinganti-lipoidal antibodies by infection of a host animal with T. pallidum(see, e.g., Perine et al., Infect. Immun., 8(5):787-790, 1973).Moreover, custom antibody production services are commercially available(see, e.g., Spring Valley Laboratories (Woodbine, Md., USA); MaineBiotechnology Services (Portland, Me., USA); CovalAb UK (Cambridge,United Kingdom); 21st Century Biochemicals (Marlboro, Mass., USA) andnumerous others). Such commercial services could be used to producelipoidal-antigen-specific polyclonal or monoclonal antibodies.

In embodiments involving antiserum containing anti-lipoidal antibodies,an antibody fraction can be isolated from the serum using well knownmethods. Commercially available kits are suitable for use in isolatingantibodies (including anti-lipoidal antibodies) from serum. Such kitsare available from, for example, Millipore (Billerica, Mass., USA),Pierce (Rockford, Ill., USA); BioRad (Hercules, Calif., USA), and manyothers. One specific non-limiting method for isolating anti-lipoidalantibodies from serum is described in Examples 2-3.

Other exemplary anchor antibodies that can be used in the disclosedcompositions and methods, include anti-cholesterol antibodies andanti-cardiolipin antibodies. Anti-cholesterol antibodies are present inhuman sera and can be isolated as described above for anti-lipoidalantibodies. Specific anti-cholesterol antibodies and/or protocols forproducing and/or isolating anti-cholesterol antibodies can be found inKruth et al. (J. Lipid Res., 42:1492-1500, 2001), Alving et al.(Biochem. Soc. Trans., 17:637-639, 1989), Swartz et al. (Proc. Natl.Acad. Sci. USA, 85:1902, 1988), Stollar et al. (Mol. Immunol.,26(1):73-79, 1989), and PCT Publication Nos. WO 00/06200, WO 97/21099,and WO 02/083100. Purified anti-cardiolipin antibodies are commerciallyavailable, for instance, from United States Biological (Swampscott,Mass., USA; e.g., Cat. No. C1375).

An anchor antibody for use in the present compositions and methodspreferably (i) does not agglutinate with other antigen-bound anchorantibodies, and (ii) does not substantially bind to (or react with) areagent (or series of reagents) used to detect an analyte of interest(such as, an anti-lipoidal antibody present in sample).

Agglutination is a process whereby multivalent antibodies form across-linked network bridged by their corresponding antigens, which mustat least two binding sites for the antibody of interest (referred to asa “multivalent antigen”). Under these circumstances, a single antibodycan bind to two different antigens and two different antibodies can bindto the same antigen. Agglutination occurs at certain concentrations ofmultivalent antibody with multivalent antigen. One exemplary way toavoid agglutination of an anchor antibody with lipoidal antigens (whichare multivalent by nature), is to use Fab (or other monovalent) antibodyfragments to anchor a lipoidal antigen. A monovalent antibody fragmentcannot bind two antigens and, thereby, cannot serve to crosslink twodifferent antigens. Methods of making monovalent antibody fragments arewell known in the art. One non-limiting method for making and isolatingFab fragments is provided in Example 5.

An alternative, non-limiting method for avoiding agglutination is toapply anchor antibodies (regardless of valency) to a solid surface (suchas, a membrane of a membrane-based immunoassay device) in the absence oflipoidal antigen; then, bind the lipoidal antigen to the immobilizedantibody. In this alternative method, the antibodies and antigens arephysically constrained from substantial crosslinking.

B. Solid Support

A solid support (or substrate) is any material which is insoluble, orcan be made insoluble by a subsequent reaction. The methods ofimmobilizing a lipoidal antigen disclosed herein can be used with anysolid support to which an anchor antibody will attach in a manner thatsubstantially resists detachment when washed with an aqueous solution,such as when contacted with a fluid sample (such as a biologicalsample). Preferred solid support embodiments for disclosed immunoassaydevices involve microporous membranes, such as nitrocellulose, nylon,polyvinylidene fluoride (PVDF), polyethersulfone, polycarbonate,polyester, cellulose acetate, mixed cellulose esters, or combinationsthereof.

The surface of a solid support may be activated by chemical processesthat cause covalent linkage of an agent (e.g., an anchor antibody oranchor antibody-lipoidal antigen complex) to the support. However, anyother suitable method may be used for immobilizing an agent (e.g., ananchor antibody or anchor antibody-lipoidal antigen complex) to a solidsupport including, without limitation, ionic interactions, hydrophobicinteractions, covalent interactions and the like. The particular forcesthat result in immobilization of an agent on a solid phase are notdeterminative for the methods and devices described herein.

A solid phase can be chosen for its intrinsic ability to attract andimmobilize an agent, such as a capture reagent. Alternatively, the solidphase can possess a factor that has the ability to attract andimmobilize an agent, such as an anchor antibody or anchorantibody-lipoidal antigen complex. The factor can include a chargedsubstance that is oppositely charged with respect to, for example, ananchor antibody or anchor antibody-lipoidal antigen complex or to acharged substance conjugated to the anchor antibody or anchorantibody-lipoidal antigen complex.

Numerous and varied solid supports are known to those in the art andinclude, without limitation, bibulous or microporous membranes (such as,nitrocellulose, nylon or PVDF), the walls of wells of a reaction tray,test tubes, polystyrene beads, magnetic beads, and microparticles (suchas latex particles). With regard to certain membrane embodiments, theporous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents, for instance,anchor antibodies or anchor antibody-lipoidal antigen complexes. Nylonpossesses similar characteristics and is also suitable. Microporousstructures are useful, as are materials with gel structure in thehydrated state.

Further examples of useful solid supports include: natural polymericcarbohydrates and their synthetically modified, cross-linked orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers which may be prepared with suitably porousstructures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

Except as otherwise physically constrained, a solid support may be usedin any suitable shape, such as films, sheets, strips, or plates, or itmay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

C. Immobilizing Anchor Antibody-Lipoidal Antigen Complexes

In some embodiments, lipoidal antigens are immobilized on a solidsupport by contacting in solution the lipoidal antigen with anchorantibodies specific for the lipoidal antigen (e.g., anti-lipoidalantibodies, anti-cholesterol antibodies, anti-lecithin antibodies,and/or anti-cardiolipin antibodies, and/or antigen-binding fragments ofany of the foregoing) to form a lipoidal antigen-anchor antibody complex(or complex). In particular embodiments, lipoidal antigen is contactedin solution with a Fab fragment specific for the lipoidal antigen (e.g.,a Fab fragment isolated from immunoglobulins in syphilitic human serumor serum from other T. pallidum-infected or -inoculated subject).

A lipoidal antigen-anchor antibody complex has a polypeptide component(i.e., an anchor antibody) and a lipid component (i.e., a lipoidalantigen). In contrast to lipids, it is well known in the art thatpolypeptides (e.g., proteins) will strongly adhere (via incompletelycharacterized interactions) to many types of solid supports and, inparticular, to membranous supports (like nitrocellulose, nylon or PVDF)(see, e.g., Harvey, Optimization of Nitrocellulose Membrane BasedImmunoassays, Keene, N. H.: Schleicher and Schuell, 1991; Wallis et al.,Ann. Rev. Microbiol., 33:413-437, 1979; Presswood, Membrane FiltrationApplications and Problems, New York, N.Y.: Marcel Dekker, 1981; Farrahet al., Proc. Natl. Acad. Sci. USA, 78:1229-1232, 1981; Batteiger etal., J. Immunol. Meth., 55:297-307, 1982; Tijssen, Practice and Theoryof Immunoassays, 8th ed, Amsterdam: The Netherlands Elsevier, 1993).Thus, via its association with a polypeptide component (e.g., anchorantibody) it is also now possible to strongly adhere (e.g., immobilize)a lipoidal antigen to a solid support (such as, nitrocellulose, nylon,or PVDF). Accordingly, the disclosed methods (and methods of making thedisclosed compositions) contemplate contacting a solid support (such asa microporous membrane) with a solution of antigen-antibody complexwherein, through such contact, the antigen-antibody complex becomessubstantially immobilized on the solid support. In some examples, ananchor antibody-lipoidal antigen complex is substantially immobilized ona solid support when no more than about 1%, no more than about 2%, nomore than about 5%, no more than about 10%, or no more than about 25% ofthe antigen-antibody complex becomes detached from the solid supportwhen the support is contacted with a fluid sample for a time sufficientfor the fluid sample to wet the solid support (e.g., for a timesufficient for a fluid sample to migrate along a membranous strip andcontact an area where a lipoidal antigen is immobilized).

To immobilize a lipoidal antigen to a solid surface, it is alsocontemplated that a solid surface (such as, nitrocellulose, nylon orPVDF) can be contacted with anchor antibody (e.g., in solution) in theabsence of lipoidal antigen. The polypeptide anchor antibody stronglyadheres to the solid surface, as discussed above, and is immobilized.Thereafter, the immobilized anchor antibody is contacted with lipoidalantigen (e.g., in solution). Specific binding of the lipoidal antigen bythe immobilized anchor antibody serves to also immobilize the antigen.As discussed above, this antigen immobilization technique can be used toavoid agglutination in circumstances where the anchor antibody ismultivalent and capable of mediating agglutination in the present of amultivalent antigen.

Under circumstances where it is desirable to detect alipoidal-antigen-binding analyte (such as, anti-lipoidal antigens) usingan immobilized lipoidal antigen, it is advantageous for the immobilizedantigen to have exposed binding sites for the analyte. Accordingly, inthe foregoing situations, amounts of anchor antibody that will notsaturate (e.g., block) the majority (or predominantly all) of theanalyte binding sites on a lipoidal antigen are used to form an anchorantibody-lipoidal antigen complex. A non-saturating amount of anchorantibody in an anchor antibody-lipoidal antigen complex is any amount ofsuch antibody that will permit an analyte (e.g., anti-lipoidalantibodies in a mobile phase) to detectably bind the lipoidal antigencomponent of the complex. In some examples, no more than about 1%, nomore than about 5%, no more than about 10%, no more than about 25%, nomore than about 30% of the available anti-lipoidal antibody bindingsites are blocked by the anchor antibody. In other examples, from 10 ngto about 1000 ng of anchor antibody in a 1 μl volume are reacted with anequal volume of lipoidal antigen prepared, for example, as described inExample 1. In particular examples, from 25 ng to about 750 ng, from 50ng to about 600 ng, from about 100 ng to about 500 ng, or from about 150ng to about 400 ng anchor antibody are used to prepare an anchorantibody-lipoidal antigen complex.

FIG. 9 illustrates one particular embodiment of a lipoidal antigen(e.g., a USR antigen) attached to a solid support (e.g., anitrocellulose membrane) via an anchor antibody (e.g., an anti-lipoidalFab). The figure further illustrates the capture by the immobilizedlipoidal antigen of an anti-lipoidal antibody from an exemplary sample(such as, a serum sample) and a relationship between a detector, thecaptured anti-lipoidal antibody, the antigen, and the anchor antibody.

Particular examples exclude anchoring a lipoidal antigen to a substrateusing an anchor antibody specific for a derivative group (such as,biotin, hexa-His, FLAG, or other epitope tag) which has been added to alipoidal antigen component with the purpose of serving as an epitope foran anchor antibody. Such examples do not exclude including derivatizedcardiolipin, derivatized lecithin and/or derivatized cholesterol ascomponents of a lipoidal antigen; however, in such examples, thederivative groups of such derivatized components do not serve asepitopes for an anchor antibody.

1. Application of Anchor Antibody-Lipoidal Antigen Complex toMicroporous Membranes

Some disclosed methods and compositions contemplate an anchorantibody-lipoidal antigen complex (also referred to as a “capturereagent”) to be attached to a microporous membrane (such asnitrocellulose, nylon or PVDF). The membrane serves to immobilize thecapture reagent and to provide a surface across or through which anapplied sample will flow or pass. Nitrocellulose (whether pure ormodified in any manner known in the art) is a preferred membrane for thedisclosed devices and methods. Nitrocellulose is thought to bindproteins by hydrogen bonding, hydrophobic interactions, and byelectrostatic mechanisms (see, e.g., Millipore Corporation, A ShortGuide Developing Immunochromatographic Test Strips, 2nd Edition, pp.1-40, 1999, available by request at (800) 645-5476).

For protein-containing capture reagents, such as an anchorantibody-lipoidal antigen complex, the dipole of the nitrate ester ofnitrocellulose is believed to interact with the strong dipole of thepeptide bonds of the protein. Salts at high concentrations, detergents,and water in an application solution may weaken and destabilizeelectrostatic interactions between a nitrocellulose membrane and aprotein to be applied to the membrane. Thus, it is preferable, althoughnot required, to use a low molarity buffer, for example, 2-10 mMphosphate, borate or carbonate buffers, to solubilize protein-containingcapture reagents for immobilization onto nitrocellulose.

The pH of an application solution may, but need not, be adjusted toincrease binding of the capture reagent to a nitrocellulose membrane.For example, the solubility of a protein-containing capture reagent inan application solution is at a minimum when the pH of the applicationsolution is within about +/−1 pH unit of the pI of theprotein-containing capture reagent.

Optionally, 1 to 5% methanol, ethanol or isopropanol may be added to anapplication solution. An application solution may be applied to amembrane manually or in an automated manner. For example, a reagentdispensing module (e.g., Matrix 1600, Kinematic Automation, Twain Harte,Calif.) may be used to apply capture reagent to a microporous membrane(such as, nitrocellulose).

Typically, blocking of a microporous membrane in the disclosed methodsand devices is not necessary. For example, proteins that are present inthe sample and other blocking agents, which may be added, e.g., to asample pad or conjugate pad of a lateral flow device, are generallysufficient to prevent an analyte from being non-specifically adsorbedonto the membrane. If optional blocking a membrane is desired for aparticular application, useful blocking agents include, for example,gelatin (0.1%-0.5%), nonfat dry milk (0.5%-2%), casein (1%-2%), BSA(1%-2%), IgG (1%-2%), PVP 8-10 kD (0.5%-1.0%), and PVA 8-10 kD(0.5%-1.0%).

V. Immunoassay Devices

The discovery herein of a method to immobilize a lipoidal antigen (suchas, a VDRL and/or USR antigen) to a solid support (such as, amicroporous membrane, like nitrocellulose, nylon or PVDF) enables theproduction of immunoassay devices for the detection oflipoidal-antigen-binding analytes (such as, anti-lipoidal antibodies inbiological samples from T. pallidum-infected subjects). In someexamples, a disclosed immunoassay device permits detection of thepresence (or absence) of anti-lipoidal antibodies in a biological samplefor diagnosis of syphilis.

A. Representative Immunoassay Device Formats and Related Information

Immunoassay devices permit the performance of relatively inexpensive,disposable, membrane-based assays for the visual identification of thepresence (or absence) of an analyte in a liquid sample. Such devices areusually formatted as freestanding dipsticks (e.g., test strips) or asdevices having some sort of housing. Typically, an immunoassay devicecan be used with as little as about 200 μl of liquid sample, anddetection of an analyte in the sample can (but need not) be completewithin 2-5 minutes. In clinical assays, the sample may be urine, blood,serum, saliva, or other body fluids. In nonclinical tests, the samplemay be a small volume of solution prepared from soil, dust, plants, orfood, and similarly applied directly to the membrane test strip. In mostinstances, no ancillary instrumentation is required to perform suchtests, and such devices easily can be used in clinics, laboratories,field locations, and the home even by inexperienced persons.

Immunoassay devices have been developed for the routine identificationor monitoring of physiological and pathological conditions (e.g.,infectious diseases, pregnancy, cancer, endocrine disorders) usingdifferent biological samples (e.g., urine, serum, plasma, blood,saliva), and for analysis of environmental samples (e.g., natural fluidsand industrial plant effluents) for instance for contamination. Many ofthese tests are based on the highly specific interactions betweenspecific binding pairs. Examples of such binding pairs includeantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. Furthermore, many ofthese tests involve devices (e.g., solid phase, lateral flow teststrips, flow-through tests) with one or more of the members of a bindingpair attached to a mobile or immobile solid phase material such as latexbeads, glass fibers, glass beads, cellulose strips or nitrocellulosemembranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).

One principle category of immunoassay is the “sandwich” assay. Ingeneral, sandwich immunoassay procedures call for mixing a sample, whichmay contain an analyte of interest (for example, anti-lipoidalantibody), with a detector reagent that specifically recognizes theanalyte, for example, gold-conjugated Protein A, gold-conjugated ProteinG, or gold-conjugated secondary antibody specific for anti-lipoidalantibody (e.g., anti-human Ab or anti-human Ab(Fc) secondary antibody).The detector reagent is mobile and typically is linked to a label oranother signaling reagent, such as dyed latex, a colloidal metal sol, ora radioisotope. This mixture is then applied to a chromatographic medium(such as a microporous or bibulous membrane, like nitrocellulose, nylonor PVDF) containing a band or zone of immobilized antigens recognized bythe analyte antibody of interest. The chromatographic medium often is inthe form of a strip that resembles a dipstick or which can beincorporated into a housing, such as in a lateral flow device orflow-through device. When the complex of the molecule to be assayed andthe detector reagent reaches the zone of the immobilized antigens on thechromatographic medium, binding occurs and the detector reagent complexis localized at the zone. This indicates the presence of the molecule tobe assayed. This technique can be used to obtain quantitative orsemi-quantitative results. Examples of sandwich immunoassays performedon test strips are described in, for example, U.S. Pat. Nos. 4,168,146and 4,366,241.

In other common forms of membrane-based immunoassays, as typified bysome home pregnancy and ovulation detection kits, a test strip (ordipstick) is “dipped” into a sample suspected of containing the subjectanalyte. Enzyme-labeled detector reagent is then added, eithersimultaneously or after an incubation period. The device next is washedand then inserted into a second solution containing a substrate for theenzyme. The enzyme label, if present, interacts with the substrate,causing the formation of colored products, which either deposit as aprecipitate onto the solid phase or produce a visible color change inthe substrate solution. EP-A 0 125 118 describes such a sandwich typedipstick immunoassay. EP-A 0 282 192 describes a dipstick device for usein competition type assays.

Flow-through type immunoassay devices were designed, in part, to obviatethe need for incubation and washing steps associated with dipstickassays. Flow-through immunoassay devices involve a capture reagent (suchas an anchor antibody-lipoidal antigen complex) bound to a porousmembrane or filter to which a liquid sample is added. As the liquidflows through the membrane, target analyte (such as, anti-lipoidalantibody) binds to the capture reagent. The addition of sample isfollowed by addition of detector reagent (such as, gold-conjugatedProtein A or gold-conjugate anti-human IgG(Fc)). Alternatively, thedetector reagent may be placed on the membrane in a manner that permitsthe detector to mix with the sample and thereby label the analyte. Thevisual detection of detector reagent provides an indication of thepresence of target analyte in the sample. Representative flow-throughimmunoassay devices are described in U.S. Pat. Nos. 4,246,339;4,277,560; 4,632,901; 4,812,293; 4,920,046; and 5,279,935; and U.S. Pat.Appl. Nos. 20030049857 and 20040241876.

Migration assay devices usually incorporate within them reagents thathave been attached to colored labels, thereby permitting visibledetection of the assay results without addition of further substances.See, for example, U.S. Pat. No. 4,770,853; PCT Publication No. WO88/08534 and European Patent No. EP-A 0 299 428.

There are a number of commercially available lateral flow type tests andpatents disclosing methods for the detection of large analytes (MWgreater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes asemiquantitative competitive immunoassay lateral flow method formeasuring plasma lipoprotein levels. This method utilizes a plurality ofcapture zones or lines containing immobilized antibodies to bind boththe labeled and free lipoprotein to give a semi-quantitative result.

U.S. Pat. No. 5,591,645 provides a chromatographic test strip with atleast two portions. The first portion includes a movable tracer and thesecond portion includes an immobilized binder capable of binding to theanalyte. Additional examples of lateral flow tests for large analytesare disclosed in the following patent documents: U.S. Pat. Nos.4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; EuropeanPatent No. 0296724; WO 97/06439; and WO 98/36278.

There are also lateral flow type tests for the detection ofsmall-analytes (MW 100-1,000 Daltons). Generally, these small analytetests involve “typical” competitive inhibition to produce negative orindirect reporting results (i.e., reduction of signal with increasinganalyte concentration), as exemplified by U.S. Pat. No. 4,703,017.However, several approaches have been developed for detecting smallanalytes using lateral flow tests that produce positive or directreporting results (i.e., increase in signal with increasing analyteconcentration). These include, for instance, U.S. Pat. Nos. 5,451,504;5,451,507; 5,798,273; and 6,001,658.

U.S. Pat. No. 5,451,504 provides a method with three specific zones(mobilization, trap and detection) each containing a different latexconjugate to yield a positive signal. The mobilization zone containslabeled antibody to bind the analyte in the sample. In the trap zone,unbound, labeled antibody is then trapped by immobilized analyte analog.The detection zone captures the labeled analyte-antibody complex.

U.S. Pat. No. 5,451,507 describes a two-zone, disconnected immunoassaymethod. The first zone has non-diffusively bound reagent that binds witha component, for example, an analyte analog bound to, or capable ofbecoming bound to, a member of a signal producing system. The secondzone binds to the component only when the analyte to be tested ispresent. The distance the component migrates into the second zone isdirectly related to the concentration of analyte.

U.S. Pat. No. 5,798,273 discloses a lateral flow device that includes acapture zone with immobilized analyte analog and one or more read-outzones to bind labeled analyte-analog.

U.S. Pat. No. 6,001,658 discloses a test strip device with a diffusible,labeled binding partner that binds with analyte, an immobilized analyte,and a detection area containing an immobilized antibody.

Devices described herein generally include a strip of absorbent material(such as a microporous membrane), which, in some instances, can be madeof different substances each joined to the other in zones, which may beabutted and/or overlapped. In some examples, the absorbent strip can befixed on a supporting non-interactive material (such as nonwovenpolyester), for example, to provide increased rigidity to the strip.Zones within each strip may differentially contain the specific bindingpartner(s) and/or other reagents required for the detection and/orquantification of the particular analyte being tested for, for example,an anti-lipoidal antibody. Thus these zones can be viewed as functionalsectors or functional regions within the test device.

In general, a fluid sample (or a sample suspended in a fluid) isintroduced to the strip at the proximal end of the strip, for instanceby dipping or spotting. A sample is collected or obtained using methodswell known to those skilled in the art. The sample containing theanti-lipoidal antibodies to be detected may be obtained from anybiological source. Examples of biological sources include blood serum,blood plasma, urine, spinal fluid, saliva, fermentation fluid, lymphfluid, tissue culture fluid and ascites fluid of a human or animal. Thesample may be diluted, purified, concentrated, filtered, dissolved,suspended or otherwise manipulated prior to immunoassay to optimize theimmunoassay results. The fluid migrates distally through all thefunctional regions of the strip. The final distribution of the fluid inthe individual functional regions depends on the adsorptive capacity andthe dimensions of the materials used.

In some embodiments, porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

Another common feature to be considered in the use of immunoassaydevices is a means to detect the formation of a complex between ananalyte (such as an anti-lipoidal antibody) and a capture reagent (suchas anchor antibody-lipoidal antigen complexes). A detector (alsoreferred to as detector reagent) serves this purpose. A detector may beintegrated into an immunoassay device (for example included in aconjugate pad, as described below), or may be applied to the device froman external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte (such as,gold-conjugated Protein A for an antibody analyte, or gold-labeledanti-human Ab(Fc) for a human antibody analyte). In other instances, adetector reagent collectively includes an unlabeled first bindingpartner specific for the analyte and a labeled second binding partnerspecific for the first binding partner and so forth. In each instance, adetector reagent specifically detects bound analyte of ananalyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent can specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

Preferably, a detector reagent for use in the disclosed methods ordevices does not substantially bind to or react with an immobilizedanchor antibody-lipoidal antigen complex. One of ordinary skill in theart can easily determine combinations of particular detector reagentsand particular anchor antibody-lipoidal antigen complexes that willsatisfy this preference for the detection of particular analytes. Forexample, for the detection of human anti-lipoidal antibodies, somenon-limiting exemplary combinations include:

Lipoidal Antigen Anchor Antibody Detector Reagent CL/L/Ch human,anti-lipoidal antigen-binding Protein A, Protein G, or fragment (e.g.,Fab) anti-human Ab(Fc) CL/L/Ch human, anti-cholesterol antigen-bindingProtein A, Protein G, or fragment (e.g., Fab) anti-human Ab(Fc) CL/L/Chhuman, anti-cardiolipin antigen-binding Protein A, Protein G, orfragment (e.g., Fab) anti-human Ab(Fc) CL/L/Ch non-human, anti-lipoidalfull-length Ab anti-human Ab (any specificity) CL/L/Ch non-human,anti-cholesterol full-length anti-human Ab (any specificity) Ab CL/L/Chnon-human, anti-cardiolipin full-length anti-human Ab (any specificity)Ab CL/L/Ch non-human (mammalian), anti-lipoidal Protein A, Protein G,anti-human antigen-binding fragment (e.g., Fab) Ab (any specificity)CL/L/Ch non-human (mammalian), Protein A, Protein G, anti-humananti-cholesterol antigen-binding Ab (any specificity) fragment (e.g.,Fab) CL/L/Ch non-human (mammalian), Protein A, Protein G, anti-humananti-cardiolipin antigen-binding Ab (any specificity) fragment (e.g.,Fab)

B. Flow-Through Device Construction and Design

A flow-through device involves a capture reagent (such as an anchorantibody-lipoidal antigen complex) immobilized on a solid support,typically, a membrane (such as, nitrocellulose, nylon, or PVDF).Characteristics of useful membrane have been previously described;however, it is useful to note that in a flow-through assay capillaryrise is not a particularly important feature of a membrane as the samplemoves vertically through the membrane rather than across it as in alateral flow assay. In a simple representative format, the membrane of aflow-through device is placed in functional or physical contact with anabsorbent layer (see, e.g., description of “absorbent pad” below), whichacts as a reservoir to draw a fluid sample through the membrane.Optionally, following immobilization of a capture reagent, any remainingprotein-binding sites on the membrane can be blocked (either before orconcurrent with sample administration) to minimize nonspecificinteractions. An exemplary physical embodiment of a flow-through deviceis shown in FIG. 10.

In operation of a flow-through device, a fluid sample (such as a bodilyfluid sample) is placed in contact with the membrane. Typically, aflow-through device also includes a sample application area (orreservoir) to receive and temporarily retain a fluid sample of a desiredvolume. The sample passes through the membrane matrix. In this process,an analyte in the sample (such as an anti-lipoidal antibody) canspecifically bind to the immobilized capture reagent (such as anchorantibody-lipoidal antigen complex). Where detection of ananalyte-capture reagent complex is desired, a detector reagent (such aslabeled Protein A, labeled Protein G, or labeled anti-human IgG(Fc)) canbe added with the sample or a solution containing a detector reagent canbe added subsequent to application of the sample. If an analyte isspecifically bound by capture reagent, a visual representativeattributable to the particular detector reagent can be observed on thesurface of the membrane. Optional wash steps can be added at any time inthe process, for instance, following application of the sample, and/orfollowing application of a detector reagent.

C. Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateralflow device is an analytical device having as its essence a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte canflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; and 6,368,876; EP0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439,each of which is incorporated by reference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials can be used, and rendered bibulous by applying asurfactant to the material), and allowed to migrate along the stripuntil the liquid comes into contact with a specific binding partner thatinteracts with an analyte in the liquid. Once the analyte interacts withthe binding partner, a signal (such as a fluorescent or otherwisevisible dye) indicates that the interaction has occurred. Multiplediscrete binding partners can be placed on the strip (for example inparallel lines) to detect multiple analytes in the liquid. The teststrips can also incorporate control indicators, which provide a signalthat the test has adequately been performed, even if a positive signalindicating the presence (or absence) of an analyte is not seen on thestrip.

The construction and design of lateral flow devices is very well knownin the art, as described, for example, in Millipore Corporation, A ShortGuide Developing Immunochromatographic Test Strips, 2nd Edition, pp.1-40, 1999, available by request at (800) 645-5476; and Schleicher &Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp.73-98, 2003, 2003, available by request at Schleicher & SchuellBioScience, Inc., 10 Optical Avenue, Keene, N. H. 03431, (603) 352-3810;both of which are incorporated herein by reference in their entireties.

Lateral flow devices have a wide variety of physical formats that areequally well known in the art. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure. FIG. 7 showsseveral examples of lateral flow devices. These examples demonstratesome of the physical embodiments that may be useful in the constructionof a lateral flow device.

The basic components of a particular embodiment of a lateral flow deviceare illustrated in FIG. 8, which shows a particular embodiment in whichan elongated housing 10 contains a bibulous lateral flow strip 12 thatextends substantially the entire length of housing 10. Lateral flowstrip 12 is divided into a proximal sample application pad 14 positionedbelow a sample introduction port 15, an intermediate test resultmembrane 16, and a distal absorbent pad 18. Flow strip 12 is interruptedby a conjugate pad 20 that contains labeled conjugate (such asgold-conjugated Protein A, gold-conjugated Protein G, gold-conjugatedanti-human Ab). A flow path along strip 12 passes from proximal pad 14,through conjugate pad 20, into test result membrane 16, for eventualcollection in absorbent pad 18. Selective binding agents (such as ananchor antibody-lipoidal antigen complex) are positioned on a proximaltest line 22 in test result membrane 16. A control line 24 is providedin test result membrane 16 slightly distal to test line 22.

In operation of the particular embodiment of a lateral flow deviceillustrated in FIG. 8, a fluid sample containing an analyte of interest,such as an anti-lipoidal antibody, is applied to the sample pad 14through the sample introduction port 15. In some examples, the samplemay be applied to the sample introduction port 15 dropwise or, lesspreferably, by dipping the end of the device containing the sampleintroduction port 15 into the sample. In other examples where a sampleis whole blood, an optional developer fluid is added to the blood sampleto cause hemolysis of the red blood cells and, in some cases, to make anappropriate dilution of the whole blood sample. From the sample pad 14,the sample passes, for instance by capillary action, to the conjugatepad 20. In the conjugate pad 20, the analyte of interest may bind (or bebound by) a mobilized or mobilizable detector reagent. For example, ananti-lipoidal antibody analyte may bind to a gold-conjugated Protein Adetector reagent contained in the conjugate pad. The analyte complexedwith the detector reagent may subsequently flow to the test resultmembrane 16 where the complex may further interact with ananalyte-specific binding partner (such as an anchor antibody-lipoidalantigen complex), which is immobilized at the proximal test line 22. Insome examples, an anti-lipoidal antibody complexed with a detectorreagent (such as, gold-conjugated Protein A, gold-conjugated Protein G,or gold-conjugated anti-human Ab) may further bind to unlabeled, anchorantibody-lipoidal antigen complexes immobilized at the proximal testline 22. The formation of the immunocomplex between anti-lipoidalantibody, labeled (e.g., gold-conjugated) detector reagent, andimmobilized anchor antibody-lipoidal antigen complex can be detected bythe appearance of a visible line at the proximal test line 22, whichresults from the accumulation of the label (e.g., gold) in the localizedregion of the proximal test line 22. The control line 24 may contain animmobilized, detector-reagent-specific binding partner, which can bindthe detector reagent in the presence or absence of the analyte. Suchbinding at the control line 24 indicates proper performance of the test,even in the absence of the analyte of interest.

In another embodiment of a lateral flow device, there may be a secondtest line located parallel or perpendicular (or in any other spatialrelationship) to test line 22 in test result membrane 16. The operationof this particular embodiment is similar to that described in theimmediately preceding paragraph with the additional considerations that(i) a second detector reagent specific for a second analyte, such as ananti-T. pallidum antibody, may also be contained in the conjugate pad,and (ii) the second test line will contain a second specific bindingpartner having affinity for a second analyte in the sample. For example,the second test line may contain immobilized treponemal antigens thatwill specifically bind anti-T. pallidum antibodies present in thesample.

Some of the materials that may be useful for the components of a lateralflow device are shown in Table 1. However, one of skill in the art willrecognize that the particular materials used in a particular lateralflow device will depend on a number of variables, including, forexample, the analyte to be detected, the sample volume, the desired flowrate and others, and can routinely select the useful materialsaccordingly.

TABLE 1 Component Useful Material Sample Pad Glass fiber Woven fibersScreen Non-woven fibers Cellulosic filters Paper Conjugate Pad Glassfiber Polyester Paper Surface modified polypropylene MembraneNitrocellulose (including pure nitrocellulose and modifiednitrocellulose) Nitrocellulose direct cast on polyester supportPolyvinylidene fluoride Nylon Absorbent Pad Cellulosic filters Paper

1. Sample Pad

The sample pad (such as sample pad 14 in FIG. 8) is an optionalcomponent of a lateral flow device that initially receives the sample,and may serve to remove particulates from the sample. Among the variousmaterials that may be used to construct a sample pad (see Table 1), acellulose sample pad may be beneficial if a large bed volume (e.g., 250μl/cm²) is a factor in a particular application. Sample pads may betreated with one or more release agents, such as buffers, salts,proteins, detergents, and surfactants. Such release agents may beuseful, for example, to promote resolubilization of conjugate-padconstituents, and to block non-specific binding sites in othercomponents of a lateral flow device, such as a nitrocellulose membrane.Representative release agents include, for example, trehalose or glucose(1%-5%), PVP or PVA (0.5%-2%), Tween 20 or Triton X-100 (0.1%-1%),casein (1%-2%), SDS (0.02%-5%), and PEG (0.02%-5%).

2. Membrane and Application Solution:

The types of membranes useful in a lateral flow device (such asnitrocellulose, nylon and PVDF), and considerations for applying acapture reagent to such membranes have been discussed previously.

3. Conjugate Pad

The conjugate pad (such as conjugate pad 20 in FIG. 8) serves to, amongother things, hold a detector reagent. In some embodiments, a detectorreagent may be applied externally, for example, from a developer bottle,in which case a lateral flow device need not contain a conjugate pad(see, for example, U.S. Pat. No. 4,740,468).

Detector reagent(s) contained in a conjugate pad is released intosolution upon application of the test sample. A conjugate pad may betreated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose. A mixture of two or more release agents may be usedin any given application. In the particular disclosed embodiment, thedetector reagent in conjugate pad 20 is labeled Protein A, Protein G, oranti-human IgG(Fc).

4. Absorbent Pad

The use of an absorbent pad 18 in a lateral flow device is optional. Theabsorbent pad acts to increase the total volume of sample that entersthe device. This increased volume can be useful, for example, to washaway unbound analyte from the membrane. Any of a variety of materials isuseful to prepare an absorbent pad, see, for example, Table 1. In somedevice embodiments, an absorbent pad can be paper (i.e., cellulosicfibers). One of skill in the art may select a paper absorbent pad on thebasis of, for example, its thickness, compressibility,manufacturability, and uniformity of bed volume. The volume uptake of anabsorbent made may be adjusted by changing the dimensions (usually thelength) of an absorbent pad.

D. Combination Devices

Each of the immunoassay devices discussed above (e.g., dipstick,flow-through device or lateral flow device) can be, in some embodiments,formatted to detect multiple analytes by the addition of secondary,tertiary or more capture areas containing capture reagents specific forthe other analytes of interest. In particular, this disclosurecontemplates immunoassay devices that concurrently detect anti-lipoidalantibody and treponemes or anti-treponemal antibodies in fluid samples(such as, human serum). Such combination devices further include atreponemal capture area involving (a) an immobilized treponemal antigencapable of being specifically bound by an anti-T. pallidum antibody, or(b) an immobilized anti-T. pallidum antibody that specifically binds amobile treponemal antigen. As used herein, a “treponemal antigen” is anantigen containing at least one antigenic determinant that specificallybinds anti-T. pallidum antibodies. Numerous treponemal antigens havebeen described in the art; see, for example, U.S. Pat. Nos. 6,479,248;6,248,331; 5,681,934; 5,578,456; 4,868,118; and 4,740,467. For instance,polypeptides of at least the following apparent molecular weights havebeen described as T. pallidum antigens: 16-20 kDa, 18 kDa, 18-23 kDa, 25kDa, 35 kDa, 37 kDa, 37-46 kDa, 38 kDa, 39 kDa, 41 kDa, 43 kDa, 44 kDa,46 kDa, 47 kDa, 58 kDa, 150 kDa; and 180 kDa (for more particulardetail, see U.S. Pat. No. 4,846,118).

Treponemal antigens and anti-T. pallidum antibodies are polypeptides;thus, when used as capture reagents, these molecules can be directlyadhered to a solid support (such as, nitrocellulose, nylon or PVDF).Nonetheless, it is contemplated that treponemal antigens or anti-T.pallidum antibodies can be immobilized (directly or indirectly) on asolid support by any available method.

A detector reagent can be used to detect the formation of a complexbetween a treponemal capture reagent and treponeme-specific analyte(such as, a treponeme, a treponemal antigen, or an anti-treponemalantibody). In some embodiments, a detector reagent (such as ananti-human Ab(Fc)) can specifically detect a bound treponeme-specificanalyte (e.g., a human anti-treponemal antibody) and a boundanti-lipoidal antibody analyte (e.g., a human anti-lipoidal antibody).In other instances, two separate detector reagents for specificdetection of a bound treponeme-specific analyte (e.g., anti-treponemalantibody or treponemal antigen) or a bound anti-lipoidal antibodyanalyte are envisioned.

The operation of an immunoassay device useful for performing concurrenttreponemal and non-treponemal tests is substantially similar to devicesdescribed elsewhere in this specification. One particular feature of acombination device is that a fluid sample applied to a sampleapplication area is able to contact (e.g., flow to or flow through) eachof an anti-lipoidal antibody capture area and to a treponemal capturearea.

VI. Kits

Disclosed herein are kits for use in detecting anti-lipoidal antibodiesin a sample (such as, a biological sample). Such kits can also be used,for example, in the diagnosis of diseases in which the presence ofanti-lipoidal antibodies is symptomatic of the disease (such as,syphilis or lupus). Certain embodiments of the disclosed kits aregenerally portable and provide a simple, rapid, and/or cost-effectiveway to detect anti-lipoidal antibodies and/or diagnose disease (such assyphilis) without the need for laboratory facilities, such as in apoint-of-care facility.

Kits include one or more immunoassay devices as disclosed herein and acarrier means, such as a box, a bag, a satchel, plastic carton (such asmolded plastic or other clear packaging), wrapper (such as, a sealed orsealable plastic, paper, or metallic wrapper), or other container. Insome examples, kit components will be enclosed in a single packagingunit, such as a box or other container, which packaging unit may havecompartments into which one or more components of the kit can be placed.In other examples, a kit includes one or more containers, for instancevials, tubes, and the like that can retain, for example, one or morebiological samples to be tested, positive and/or negative controlsamples or solutions (such as, a positive control serum containinganti-lipoidal or treponemal antibodies), diluents (such as, phosphatebuffers, or saline buffers), detector reagents (e.g., for externalapplication to a kit device), substrate reagents for visualization ofdetector reagent enzymes (such as, 5-bromo-4-chloro-3-indolyl phosphate,nitroblue tetrazoliurn in dimethyl formamide), and/or wash solutions(such as, Tris buffers, saline buffer, or distilled water).

Other kit embodiments include syringes, finger-prick devices, alcoholswabs, gauze squares, cotton balls, bandages, latex gloves, incubationtrays with variable numbers of troughs, adhesive plate sealers, datareporting sheets, which may be useful for handling, collecting and/orprocessing a biological sample. Kits may also optionally containimplements useful for introducing samples into a sample chamber of animmunoassay device, including, for example, droppers, Dispo-pipettes,capillary tubes, rubber bulbs (e.g., for capillary tubes), and the like.Still other kit embodiments may include disposal means for discarding aused immunoassay device and/or other items used with the device (such aspatient samples, etc.). Such disposal means can include, withoutlimitation, containers that are capable of containing leakage fromdiscarded materials, such as plastic, metal or other impermeable bags,boxes or containers.

In some examples, a disclosed kit will include instructions for the useof an immunoassay device. The instructions may provide direction on howto apply sample to the test device, the amount of time necessary oradvisable to wait for results to develop, and details on how to read andinterpret the results of the test. Such instructions may also includestandards, such as standard tables, graphs, or pictures for comparisonof the results of a test. These standards may optionally include theinformation necessary to quantify analyte using the test device, such asa standard curve relating intensity of signal or number of signal linesto an amount of analyte therefore present in the sample.

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Preparation of a USR Lipoidal Antigen

This Example describes the preparation of an exemplar lipoidal antigen,a USR antigen, which can be attached to a solid support, such asnitrocellulose, using the disclosed methods. Approximately 100 ml of aUSR antigen can be prepared as described in this Example; however, thoseof ordinary skill in the art will appreciate that this describedprotocol can be scaled upward or downward to produce more or less,respectively, USR antigen. Moreover, unless expressly stated otherwise,all method steps, reactions, etc. in this and the following exampleswere performed at room temperature (e.g., from about 20° C. to about 25°C.).

Eight (8) ml of VDRL buffered saline (10 grams NaCl, 0.5 mlformaldehyde, 0.093 grams disodium hydrogen phosphate, and 0.170 gramspotassium dihydrogen phosphate in 1 liter of distilled water) was addedto a 250 ml round, glass-stoppered bottle. Over a period of 40 to 60seconds, 10 ml VDRL antigen (0.9% cholesterol, 0.03% bovine heartcardiolipin, and about 0.21% lethicin in ethanol) was added directlyonto the buffered saline with continuous rotation of the bottle.Rotation of the bottle was continued for approximately 10 secondsfollowing the addition of the VDRL antigen. Then, 82 ml VDRL bufferedsaline was added to the reaction mixture. With the top placed on thebottle, the bottle was shaken from bottom to top and back approximately30 times in 10 seconds. Approximately 19 ml aliquots of the shakenmixture were centrifuged in stainless steel tubes in an angle centrifuge(Sorvall SS-3) at room temperature at a relative centrifugal force ofapproximately 2000×g for 15 minutes (measured from when the centrifugereached the desired speed). The supernatant fluid was carefully decantedby inverting the tube away from the side containing the sedimentedmaterial. After removing the supernatant, the centrifuge tube wasinverted and wiped with cotton gauze without disturbing the sediment.The sediment was then resuspended with gentle shaking in a volume ofresuspending solution (3.72% EDTA, 40% choline chloride (v/v), phosphatebuffered saline pH 6.9±0.1) equal to that of the antigen suspensioncentrifuged (in this case, 19 ml). All resuspended sediments wererecombined in a glass-stoppered bottle and gently swirled to mix to formthe antigen preparation. The antigen preparation was then placed in arefrigerator (from about 2° C. to about 8° C.) for approximately oneweek to stabilize.

Example 2 Ammonium Sulfate Fractionation of T. pallidum-Infected HumanSerum

This Example describes one method for separating animmunoglobulin-containing fraction from serum. The method of thisExample (as well as other immunoglobulin isolation methods that arecommonly known in the art) can be used to partially purifyimmunoglobulins from any source of blood serum, including serum fromnon-human subjects (such as, rabbits).

Serum from human patients infected with T. pallidum (referred tothroughout this and the following examples as “hyperimmune antiserum”)was obtained from Biomedical Resources (Hatboro, Pa.). A desired volumeof hyperimmune antiserum was placed in a container that held more thantwice the volume of serum. With stirring, an amount of 70% saturatedammonium sulfate equal to the volume of serum was slowly added dropwiseto the serum. This mixture was stirred for approximately 4 hours at roomtemperature prior to centrifugation at approximately 3000 rcf for 30minutes. Following centrifugation, the supernatant was discarded and theprecipitated immunoglobulin-containing fraction was resuspended in avolume of distilled water equal to the original volume of serum. Then,an equal amount of 70% saturated ammonium sulfate solution was addeddropwise to the resuspended immunoglobulin-containing fraction withstirring, the mixture was centrifuged, the supernatant decanted, and theprecipitate resuspended as above. This procedure was then repeated onemore time. The final immunoglobulin-containing precipitate wasresuspended with distilled water to one-tenth the original serum volume.The resuspended immunoglobulin-containing fraction was dialyzed againstthree 100× volumes of 0.85% sodium chloride pH 8.0 overnight at 2-8° C.Total protein concentration in the dialysis retentate was determined bythe Bio-Rad™ Bradford Protein Assay according to the manufacturer'sinstructions.

Example 3 Purification of IgG Using a Protein A Affinity Column

IgGs were isolated from the immunoglobulin-containing fraction ofExample 2 using Protein A agarose chromatography. As is known in theart, Protein A specifically binds to the Fc region of IgG from a varietyof mammalian species.

A buffer reservoir containing 1000 ml of 20 mM sodium phosphate buffer,pH 7.4 (“phosphate buffer”) was connected to a 10 ml Protein A SepharoseCL-4B column (Pharmacia). The column was washed with 200 ml of thephosphate buffer, and the pH of the eluant measured. The column was notloaded with the immunoglobulin-containing sample until the pH of theeluant was in the range of 7.3±0.2.

After disconnecting the buffer reservoir, the immunoglobulin-containingdialysis retentate from Example 2 was applied to the column taking carenot to disturb the surface of the column matrix. Then, 20 ml ofphosphate buffer was added to the column, the buffer reservoir wasreattached to the column, and fractions were collected using a fractioncollector loaded with 80−12×125 mm diameter tubes. The absorbance at 280nm of the column eluant was monitored. When the absorbance at 280 nm ofthe eluant returned to baseline (indicating the substantially completeelution of non-specific protein), a second buffer reservoir containing500 ml of 100 mM glycine pH 2.7 was connected to the column. Again,fractions were collected while monitoring the absorbance of the eluantat 280 nm. All fractions eluted in the glycine buffer having anabsorbance_(280nm) of 0.1 or greater were combined. Such fractionsincluded proteins specifically bound by Protein A, i.e., IgGs, that werepresent in the T. pallidum-infected human serum. The pH of the combinedsolution was adjusted to 7.2±0.2 using 2.0 M Tris buffer, and totalprotein concentration was determined as described previously.

Example 4 Specific Binding of Protein A-Purified IgG to USR Antigen inSolution

The endpoint titer of the Protein A-purified IgGs obtained as describedin Example 3 was determined. Serial dilutions of the IgG sample (from1:2 to 1:512) in 0.9% saline were made. Fifty (50) μl of each dilutionwas placed in each of 10 circles (14 mm) on a 2×3 inches glass slide.

The USR antigen, prepared as described in Example 1, was gentlyresuspended and drawn into a dispensing needle and syringe in a verticalposition. Several drops were dispensed and discarded to clear the needleof air. Then, 1 free-falling drop (approximately 22 μL) of antigensuspension was added to each circle containing IgG. The slide was placedon the mechanical rotator for 4 minutes at 180±2 rpm. When performingthis test in a dry climate where evaporation may be a problem, it may beadvantageous to place the slides under a moist humidifying cover duringthe rotation step. Immediately after rotation, the slide was viewedmicroscopically using 10× oculars and a 10× objective to determine inwhich reactions flocculation occurred. The IgG titer was determined tobe the highest dilution that gives a weak (or weakly) positive reaction.

An original volume of Protein A-purified IgG sample was diluted in afirst dilution volume of phosphate buffered saline pH 7.2±0.2 (“PBS”) toan effective antibody titer of 1:2048. Thus, in one situation where theendpoint titer of the sample was 1:32, one volume of sample was dilutedwith 64 volumes of phosphate buffered saline pH 7.2±0.2 (“PBS”) (Inother examples, a serum having a 1:16 endpoint titer will be dilutedwith 128 volumes PBS, and a serum having a 1:64 endpoint titer will bediluted with 32 volumes PBS, etc.). Then, a volume of USR micelle(prepared as described in Example 1) equal to the original volume ofpurified IgG was added to the diluted IgG sample with gentle mixing. Themixture was allowed to settle at 2-8° C. for several days until thesupernatant cleared. Then, the supernatant was removed and replaced witha volume of PBS equal to the first dilution volume (e.g., 64 volumes ofPBS in the above-described situation). The foregoing steps were repeateduntil the supernatant was clear. After removing the final supernatant,the volume of precipitated material (containing USR micelle-antibody(IgG) complexes; also referred to as IgG-coated micelles) was measuredwith a cylinder, and the protein concentration of the IgG-coated micellewas determined by the Bradford method.

Example 5 Preparation and Isolation of Fab Fragments

Protein A-purified IgG (prepared as described in Example 3) wasconcentrated to approximately 20 mg/ml using a Centricon™ centrifugalfilter (Millipore) and dialyzed against 2×1000 ml Sample Buffer (20 mMsodium phosphate, 10 mM EDTA, pH 7.0).

Papain immobilized on 6% cross-linked beaded agarose in a 50% slurry in50% glycerol, 0.1 M sodium acetate (pH 4.4) and 0.05% sodium azide(Pierce) was mixed by inversion or gentle shaking to obtain an evensuspension. Then, 2.5 ml of the slurry was added to a glass test tube orother suitable reaction vessel. To equilibrate the gel, 20 ml of freshlyprepared Digestion Buffer (20 mM sodium phosphate, 10 mM EDTA, 20 mMcysteine-HCl, pH 7.0) was added the slurry. Then, the gel was separatedfrom the buffer by centrifugation, and this procedure was repeated withanother 20 ml of Digestion Buffer. The equilibrated gel was resuspendedin 2.5 ml of Digestion Buffer.

Protein A-purified IgG (2.5 ml of a 10 mg/ml solution) was diluted with2.5 ml of Digestion Buffer. The IgG solution was add to the tube orvessel containing the immobilized papain, and the mixture was incubatedfrom five hours to overnight in a shaker water bath at 37° C. at highspeed. A constant mixing of the gel was maintained during theincubation.

Following the incubation, 7.5 ml of 10 mM Tris-HCl, pH 7.5 was added tothe reaction mixture, and mixture was separated by centrifugation at 300rpm for 25 minutes. The supernatant (which contains immunoglobulinfragments) was removed from the sedimented immobilized papain.

The supernatant was run on a Protein A column as described in Example 3.As shown in FIG. 1, two protein peaks were recovered. Peak I, which didnot specifically bind Protein A, was the Fab fraction of immunoglobulinfragments, and Peak II, which did specifically bind Protein A, was theFc fraction. The protein concentrations of Fab and Fc fractions weredetermined as previously described.

Example 6 Polyacrylamide SDS Gradient Gel Analysis ofImmunglobulin-Containing Samples

This Example illustrates the protein content of various samples producedin Examples 2, 3 and 5. A Bio-Rad™ pre-cast 10-20% linear polyacrylamidegradient gel with 4% stacking gel was used to separate, by size, theprotein bands present in the ammonium sulfate immunoglobulin fraction(see Example 2), Protein A peaks I and II (see Example 3), ProteinA-purified IgG before and after papain digestion (see Example 5), andIgG peaks I (Fab fraction) and II (Fc fraction) after papain digestion(see Example 5).

Samples (2.5 μg/μl) were diluted 1:1 in Laemmli sample buffer (62.5 mMTris-HCl, 2% SDS, pH 6.8, 2% SDS and 25% glycerol (with noβ-mercaptoethanol)). Samples were heated and loaded in the respectivewells. The tank buffer was Tris-glycine-SDS (25 mM Tris-HCl, pH 8.3, 192mM glycine, 0.1% SDS). The gel was run for 45 minutes at 200 volts.Following three washes with H₂O, the gels were stained for 30 minuteswith Pierce Gel Code Blue™ and then cleared by repeated washing withwater.

As shown in FIG. 2, the ammonium sulfate-precipitated fraction of humanhyperimmune (syphilitic) serum (lane 2) contained a mixture ofpredominantly high molecular weight protein bands. As shown in FIG. 2,lane 4, a relatively pure IgG fraction was bound by and eluted from aProtein A column. Papain digestion of the Protein A-purified IgGproduced a mixture of proteins (lane 6), which were separable into twoprotein populations by fractionation over a Protein A column. FIG. 2,lane 7 shows that one predominant papain-digested IgG fragment ofapproximately 54 kD was not retained by the Protein A column. Thisapparent molecular weight is the expected size of Fab fragments. Therewere substantially no higher molecular weight protein bands(representing, for example, Fab₂ or Fc fragments or undigested IgG)observed in lane 7. Proteins having the expected molecular weights of Fcfragments and undigested IgG were retained by the Protein A column (lane8).

Example 7 Protein A Colloidal Gold Conjugate does not Detect FabFragments

One (1) μl of the papain digestion fractions of IgG (prepared asdescribed in Example 5) were spotted on a 5 mm×4 cm nitrocellulosemembrane and dried overnight. One hundred (100) μl of 1% casein in 10 mMphosphate buffer with 0.25M sodium chloride, pH 7.4 were place intocorresponding wells of a microtiter plate. Two (2) μl of Protein Aconjugated to colloidal gold were mixed into each well and thecorresponding strips were placed into each well.

As shown in FIG. 3, colloidal gold Protein A conjugate detected the IgGand Fc fractions but not the Fab fractions.

Example 8 Determination of a Useful Amount of Fab to Attach to USRMicelle

Fab fragments prepared as described in Example 5 were diluted in PBS to1000 Ξg/μl, 100 μg/μl, 10 μg/μl, 1 μg/μl, 100 ng/μl and 10 ng/μl. EachFab fraction dilution was mixed with an equal volume of a USR micellesolution prepared as described in Example 1. One (1) μl of this Fab-USRmicelle mixture was spotted on a 5 mm×4 cm nitrocellulose membrane anddried overnight at room temperature.

A human antiserum (which was reactive in the RPR test at a 1:64dilution) was diluted 1:400 in a buffer consisting of 1% casein, 10 mMphosphate, 0.25M sodium chloride pH 7.4. A similarly dilutedRPR-non-reactive human serum was used as a negative control. One hundred(100) μl antiserum (or non-reactive serum) and 2 μl colloidal goldProtein A were combined in each of several wells of a microtiter plate.A single nitrocellulose strips containing dried Fab-reacted lipoidalantigen was placed into each well at room temperature for a timesufficient for the components placed in the wells to migrate along themembrane to the location where the Fab-USR micelle mixture was spotted.

As shown in FIG. 4, nitrocellulose strips having 1000, 10 or 0.1 μg Fabbound to USR antigen gave a positive reaction with RPR-reactive (R)human serum. No reaction was observed for any of the samples containingRPR-non-reactive (NR) serum.

This Example demonstrates that, at least, from about 100 ng to about 1mg Fab fragments (such as from about 100 ng to about 450 ng) can bereacted with a USR antigen preparation (see Example 1) to facilitateattachment of the lipoidal antigen to a nitrocellulose membrane withoutsubstantial adverse effect on the reactivity of the USR antigen withserum anti-lipoidal antibodies in an immunoassay assay (such as, teststrips, and flow-through and/or lateral flow devices). One non-limitinguseful amount of Fab fragment to use as described in this Example isabout 10 μg.

Example 9 Determination of a Useful pH for Conjugation of AffinityPurified Rabbit Anti-Human IgG (Fc) to Colloidal Gold

This Example illustrates a representative method for determining auseful pH for conjugating affinity purified rabbit anti-human IgG (Fc)(Rockland, Gilbertsville, Pa.) with colloidal gold. The rabbitanti-human IgG (Fc) specifically binds only the Fc portion of human IgG,and does not bind to the Fab or other non-Fc regions of a human IgG.Antibodies with this specificity may also be referred to as “anti-humanFc.” Colloidal gold preparations such as those described in this andother examples can be used as detector reagents in disclosedimmunoassays (including, for example, test strips, and flow-throughand/or lateral flow devices).

Approximately 25 ml of 10 mM phosphate buffer was placed in a 50 mlbeaker, and adjusted to pH 5.0 with 0.2 M phosphoric acid. Two 0.5 mlaliquots of the buffer at pH 5.0 were transferred to two 12×75 mm testtubes, one labeled “test” and the other labeled “control.” Then, the pHof the phosphate buffer remaining in the beaker was sequentiallyadjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 using0.2 M potassium carbonate. At each pH, two 0.5 ml aliquots weretransferred to a “test” and “control” test tube as described for the pH5.0 sample. Then, 30 μg of affinity purified rabbit anti-human IgG (Fc)was added to each of the “test” and “control” tubes, and the tubecontents were mixed well. Approximately 25 ml of 40 nm colloidal gold(1% solution) (British Biocell International, London, England) wasplaced in a separate 50 ml beaker, and a series of “control” and “test”colloidal gold samples at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, and 10.0 was produced as described above.

One (1) ml of colloidal gold at each pH was added to the “test” and“control” solutions having the corresponding pH. The gold/rabbitanti-human IgG (Fc) solutions were mixed well, and incubated for 20minutes at room temperature. Then, 200 μl of 2M NaCl was added to theset of tubes labeled “test” and 200 μl of distilled water was added tothe set of tubes labeled “control.” The contents of both sets of tubeswere allowed to incubate for 30 minutes at room temperature.

The optical density at 580 nm (OD₅₈₀) of each “test” sample was readagainst the corresponding “control” sample. The pH of the sample withthe lowest OD₅₈₀ was determined to be a favorable pH for formation of agold conjugate preparation. Colloidal gold particles have a negativelycharged surface due to the layer of negative ions adsorbed onto the goldparticle surface during the manufacturing process. Proteins, such as IgGwill be attracted to negatively charged gold particles through ionic,hydrophobic, and dative interactions. These interactions are believed tounderlie the formation of colloidal gold-protein conjugates. At the pIof a protein conjugated to a gold particle (i.e., the pH where theprotein has a net zero charge), the conjugate will be the most stable.

The addition of NaCl to unconjugated colloidal gold particles willdisrupt the layer of negatively charged ions adsorbed to the gold'ssurface. As a result, the gold particle will dissociate and ultimatelyrelease gold ions (i.e., Au⁺) into solution. The free gold ions may bemeasured at OD₅₈₀. In contrast, a protein-gold conjugate is resistant todisruption by NaCl at the pI of the protein component of the conjugate.Accordingly, the pH of the sample with the lowest OD₅₈₀ is a useful pHat which to perform gold conjugation reactions to obtain a stable goldconjugate.

As shown in FIG. 5, a useful pH for conjugation of 30 μg rabbitanti-human IgG (Fc) with to 10 μg colloidal gold (i.e., 1 ml of a 1%solution) is approximately pH 9.5. The method described in this Exampleis broadly applicable to any number of other proteins that may beconjugated to gold for use in a disclosed immunoassay (such as, ProteinA or Protein G). In addition, it is believed that the describedreactions are scalable to other amounts of protein and gold conjugate.

Example 10 Determination of a Useful Protein Concentration for ColloidalGold Conjugation

This Example illustrates an exemplary method for determining a usefulprotein concentration for conjugation reactions with colloidal gold.Colloidal gold preparations such as those described in this and otherexamples can be used as detector reagents in disclosed immunoassays(including, for example, test strips, and flow-through and/or lateralflow devices).

One-half (0.5) ml of 10 mM phosphate buffer (adjusted to the pH givingthe lowest OD₅₈₀ as described in Example 9) was added to 2 sets of 5tubes. One set of tubes was labeled “test” and the other set was labeled“control.” Thirty (30) μg, 20 μg, 10 μg, 5 μg, or 2.5 μg of rabbitanti-human IgG (Fc) was then added to each in the series of tubes, andmixed well. One (1) ml of 40 nm colloidal gold (1% solution adjusted tothe same pH as the phosphate buffer) was added to each tube. The sampleswere incubated for 20 minutes at room temperature. Then, 200 μl of 2MNaCl was added to the samples labeled “test” and 200 μl of distilledwater was added to the samples labeled “control.” Following incubationfor 30 minutes at room temperature, the OD₅₈₀ of the test samples wereread against the corresponding control samples. The lowest concentrationof rabbit anti-human IgG (Fc) producing the lowest OD₅₈₀ indicates auseful concentration (or concentration range) for formation of aprotein-gold conjugate preparation.

In this Example, the lowest concentration of rabbit anti-human IgG (Fc)producing the lowest OD₅₈₀ represents the concentration where a usefulamount of rabbit anti-human IgG (Fc) was associated with gold particles.Higher concentrations of rabbit anti-human IgG (Fc), although useful,are less preferred because excess rabbit anti-human IgG (Fc) may formweaker associations with the gold particles, for example, by layeringupon a layer of rabbit anti-human IgG (Fc) molecules that previouslyassociated with the gold particles.

As shown in FIG. 6, approximately 30 μg is a useful amount of rabbitanti-human IgG (Fc) to conjugate to 10 μg colloidal gold (i.e., 1 ml ofa 1% solution). The method described in this Example is broadlyapplicable to any number of other proteins that may be conjugated togold for use in a disclosed immunoassay (such as, Protein A or ProteinG). In addition, it is believed that the described reactions arescalable to other amounts of protein and gold conjugate.

A. Preparation of a Gold Conjugate “Mini-preparation”

A rabbit anti-human Fc gold conjugate mini preparation was prepared byadding 5 ml of 10 mM borate buffer to an amount of rabbit anti-human IgG(Fc) necessary to achieve a useful protein concentration (such as, 30μg) as described above. This protein solution was then adjusted to auseful pH (such as, pH 9.5) as also described above. Ten (10) ml of 40nm colloidal gold (1% solution adjusted to the useful pH) was added tothe pH-adjusted protein solution with thorough mixing. The mixture wasincubated for 20 minutes at room temperature. Then, 1.6 ml of 10% caseinwas added to a final concentration of 1% casein, and the incubationcontinued an additional 20 minutes at room temperature. The reactionmixture was centrifuged at 6500×g for 10 minutes, and the supernatantwas removed and discarded. The pellet was resuspended in 0.5 ml ofresuspension buffer (150 mM NaCl, 20 mM Trizma base, 10% sucrose, 5%Trehalose, 0.1% casein, 0.05% sodium azide).

The resuspended pellet containing gold conjugated rabbit anti-human IgG(Fc) may be used for a variety of purposes, including withoutlimitation, as a detector reagent for human anti-lipoidal antibodies ina lateral flow device.

Example 11 Detection of Anti-Lipoidal Antibodies in Human Serum

This Example demonstrates that anti-lipoidal antibodies in syphiliticserum may be detected by using a nitrocellulose immobilized Fab-USRantigen complex capture reagent in concert with a mobile rabbitanti-human IgG (Fc) or Protein A gold conjugate detector reagent.

One (1) μl of the Fab-USR antigen complex was applied to separatenitrocellulose membranes and allowed to dry overnight at roomtemperature as described in Example 8. A colloidal gold conjugate wasprepared as described in Example 10. Reactive human syphilitic sera anda non-reactive (non-syphilitic) human serum were diluted 1:100, 1:200 or1:400 in 1% casein, 10 mM phosphate, 0.5M sodium chloride pH 7.4. Onehundred (100) μl of each serum dilution was placed in an appropriatenumber of separate wells of a microtitre plate. Two (2) μl ofgold-conjugated Protein A (prepared in a manner analogous to Example 10)was then added to each well. One nitrocellulose strip containingimmobilized Fab-USR antigen complex capture reagent was then placed intoeach well containing the antibody and detector reagent solutions. Thesolution in the wells flowed up the strip by capillary action.

As shown in Table 2, many of the sixty reactive human syphilitic serareacted with the immobilized Fab-USR antigen preparations. A positivereaction was characterized by a visible dot of various shades ofintensity, from very faint (VF), faint (F), weak (W) and strong (S). Novisible reaction was indicated by “N.” There was no reaction observedfor the non-reactive (non-syphilitic) human serum at any tested serumdilution.

TABLE 2 Reactivity of Immobilized Lipoidal Antibody with HumanSyphilitic Sera Serum Dilutions Serum No. RPR Titer^(a) 100 200 400 1 R4VF VF N 2 R8 N VF F 3 R2 N N N 4 R16 VF N W 5 R32 S S S 6 NR N N N 7 R64VF F W 8 R128 S S S 9 R4 VF VF N 10 R64 W W W 11 R256 S S S 12 NR N N N13 R512 S S S 14 R16 VF W W 15 R8 W W F 16 R1024 S S S 17 NR N N N 18R512 S S S 19 R64 F W W 20 R256 S S S 21 R8 VF VF F 22 R1024 S S S 23 NRN N N 24 R32 W S S 25 R16 VF F W 26 R8 W W W 27 R32 W W S 28 R4 N N VF29 NR N N N 30 R1024 S S S 31 R32 S S S 32 R16 VF W W 33 NR N N N 34 NRN N N 35 R32 W W S 36 R64 F W W 37 R8 VF F W 38 NR N N N 39 R4 N N VF 40R32 W S S 41 R8 F W W 42 R128 W S S 43 R2 N N N 44 R512 S S S 45 R16 VFW W 46 R1024 S S S 47 NR N N N 48 R64 VF F W 49 R512 S S S 50 NR N N N51 R256 S S S 52 R4 VF F N 53 R128 S S S 54 R32 W W S 55 R2 N N N 56R256 S S S 57 NR N N N 58 R64 W W S 59 R8 W W F 60 R128 S S S ^(a)RPRtiter is highest dilution of reactive syphilitic serum which gives avisible reaction in the standard rapid plasma reagin (RPR) test.

Example 12 Simultaneous Detection of Non-Treponemal and TreponemalAntibodies in Human Serum or Plasma

This Example demonstrates a flow-through test device that allows thesimultaneous detection in a biological sample (such as, human serum orplasma) of antibodies specific for treponemal or non-treponemal(lipoidal) antigens. Such device is useful, for instance, in thediagnosis of syphilis in a subject.

The flow-through test device used in this Example included a membranespotted with recombinant treponemal antigen, VDRL antigen (immobilizedas described in the foregoing Examples) and a control. The prototypedevice was provided by Span Diagnostics Ltd. (173-B, New IndustrialEstate, Udhna, Surat-394 210, India). The recombinant treponemalantigen, immobilized VDRL antigen, and control were arranged in atriangular configuration along the edges of the membrane (see, e.g.,FIG. 10). The studies were performed with the test device resting on ahorizontal surface to ensure equal distribution of the reagents duringthe test.

One hundred (100) μl of wash buffer was added to the center of the testdevice and allowed to soak in for at least 30 seconds. The wash bufferwas void of organic solvents and detergents. One hundred (100) μl of ahuman serum or plasma was then added to the center of the test devicemembrane and allowed to react with the surface for at least 30 seconds.If the samples were to be kept for a short period of time, the sampleswere stored at 2-8° C. For longer storage, the specimens were stored at−20° C. or lower. Prior to assaying previously frozen samples, thesamples were completely thawed, gently mixed and subjected tocentrifugation at 2,000×g for 10 minutes. The resulting clearsupernatant was then tested. The test device was subsequently washedwith wash buffer (150 μl for at least 30 seconds, which allowed forsolution absorption). To determine if antibodies specific for treponemaland/or non-treponemal (lipoidal) antigen(s) were present in the sample,200 μl of SIGNAL REAGENT (Colloidal Gold Protein A Reagent) was addedand allowed to soak in. For the earliest interpretation, results wereread after about 2 minutes. For a final interpretation the results wereread after about 10 minutes.

If a colored spot appeared only in the control area, then the sample wasconsidered to be non-reactive for antibodies specific for eithertreponemal or non-treponemal (lipoidal) antigens. Reactivity in thecontrol area alone indicated that the device was functioning properly,and further indicated a negative diagnosis for syphilis (or T. palliduminfection) for the subject from which the sample was obtained. If thecontrol spot and either or both of the treponemal and/or non-treponemalantigen spots were reactive (i.e., turned color), a positive diagnosisof syphilis (or T. pallidum infection) in the subject was furtherconsidered (as discussed below in more detail). If, upon completion ofthe test, none of the control, treponemal antigen or non-treponemalantigen spots appeared reactive, then the test was considered invalid.

Table 3 illustrates the results obtaining from processing 150 samplesobtained from healthy donors that had not previously been infected withT. pallidum.

TABLE 3 Flow-Through Test Sample VDRL TPHA Treponemal Non-treponemal QtyTest Test Spot Spot 150 +ve: 2 −ve: 150 −ve: 150 +ve: 2 −ve: 148 −ve:148The VDRL and TPHA tests are solution-based tests for detecting insubject samples antibodies specific for non-treponemal (lipoidal)antigens and treponemal antigens, respectively. As shown in Table 3, themembrane-based (flow-through) test for the detection of each type ofantibody in patient samples provided the identical result as thesolution-based test for the corresponding antibody. Advantageously, thedual-detection flow-through test allowed both anti-treponemal andanti-non-treponemal (anti-lipoidal) antibodies to be detected in asingle assay. Only two samples (1.3%) from a healthy subject showed apositive reaction with the VDRL test and the non-treponemal (lipoidal)antigen of the flow-through device. Thus, both tests had identical andlow incidence of false positive results and identical and no incidenceof false negative results when testing serum from subjects not infectedwith T. pallidum.

The test device was then used to analyze serum from nine patients knownto be (or have been) infected with T. pallidum (serum from such patientsis also referred to as syphilitic serum). As demonstrated in Table 4A,the membrane-based (flow-through) assay provided identical results asthe solution-based tests for each sample; that is, each sample had thesame reactivity in the solution-based VDRL test as for themembrane-bound non-treponemal antigen, and in the solution-based TPHAtest as for the membrane-bound treponemal antigen. As described abovefor the “healthy” subject serum samples, the advantage of theflow-through test was the ability to use a single assay tosimultaneously test patient serum for reactivity to treponemal andnon-treponemal (lipoidal) antigens.

TABLE 4A Flow-through Sample VDRL TPHA Treponemal Non-treponemal No.Test Test Control Antigen Antigen 1 + + + + + 2 + + + + + 3 + + + + +4 + + + + + 5 + + + + + 6 + + + + + 7 + − + − + 8 + − + − + 9 + − + − +

To compare the sensitivity of the solution-based VDRL and TPHA assays tothe flow-through test device, serum from sample 6 above was selected,serially diluted, and each dilution tested as above. As demonstrated inTable 4B, the sensitivity of the flow-through device for simultaneousdetection of anti-non-treponemal (anti-lipoidal) and anti-treponemalantibodies in syphilitic serum was similar to the single detectionsolution assay for each type of the antibody.

TABLE 4B Flow-through VDRL TPHA Non- Dilution Test Test ControlTreponemal treponemal 1:10 + + + + + 1:20 +/− + + + − 1:40 − + + + −1:80 − +/− + + − 1:160 − − + +/− − 1:320 − − + − − 1:640 − − + − −

Forty-two (42) patient samples were screened for anti-treponemal andanti-non-treponemal (anti-lipoidal) antibodies using the dual-detectionflow-through device and the TPHA and RPR solution-based assays. Intraditional testing for syphilis, patient serum often is first screenedfor reactivity with a non-treponemal (lipoidal antigen) antigen, forexample, using the RPR test. The RPR test typically is performed in alaboratory and may take several days to complete; meanwhile, the testedpatient is released from the testing facility. If the RPR test ispositive, a subsequent test to determine reactivity of patient serumwith treponemal antigen(s) (such as, the TPHA test) would be recommendedfor a positive diagnosis of syphilis. Often is it difficult to recall apatient to perform a subsequent TPHA test, which test also typically isperformed in a laboratory and may take days to complete. Testing patientsamples only for reactivity to treponemal antigen(s) (e.g., using theTPHA test) also has limitations because once a patient has been infectedwith T. pallidum (i.e., has had syphilis), his/her titer foranti-treponemal antibodies typically stays high even after successfultreatment. Hence, a positive test for treponemal antigen alone may notbe fully informative.

The performance characteristics of the flow-through device as comparedto the TPHA or RPR solution-based assays in the testing of a populationof 42 patients are presented in Table 5A and 5B, respectively.

TABLE 5A TPHA Treponemal Spot + − Total + 20 4 24 − 1 17 18 21 21 42

The sensitivity of the flow-through device for detecting anti-treponemalantibodies was 95% and the specificity was 81%.

TABLE 5B Non-Treponemal RPR Spot + − Total + 13 0 13 − 1 28 29 14 28 42The sensitivity of the flow-through device for detectinganti-non-treponemal (anti-lipoidal) antibodies was 93% and thespecificity was 100%.

Of 42 patients screened, 13 patients were found to be both RPR- andnon-treponemal spot-positive (Table 5B, top left number), and 20 werefound to be both TPHA- and treponemal spot-positive (Table 5A, top leftnumber). All 13 patients who were both RPR- and non-treponemalspot-positive were also TPHA- and treponemal spot-positive. Thus, basedon the results of the flow-through device alone, these 13 patients wouldhave been treated for syphilis immediately with no need for subsequenttesting.

In the use of the flow-through test device described in this Example,one, non-limiting set of clinical management recommendations are:

Non- Treponemal treponemal Spot Spot Clinical Recommendation − − Noaction to be taken; repeat flow-through test after about 3 months,especially in populations at high-risk for syphilis − + No syphilistreatment; investigate alternative causes for elevated anti-lipoidalantibody titer (e.g., lupus) + + Take blood for quantitative RPR testand obtain baseline measurement; treat patient for syphilis; and repeatquantitative RPR test in 6 months to determine efficacy of treatment + −No syphilis treatment; repeat flow-through test after about 3 months todetect active infection

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used and itis intended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

1. An immunoassay device comprising a microporous substrate having ananti-lipoidal antibody capture area, comprising: (a) an anchor antibodyimmobilized on the substrate; and (b) a lipoidal antigen, comprisingcardiolipin, lecithin, and cholesterol, wherein the immobilized anchorantibody is specifically bound to one or more of the cardiolipin,lecithin or cholesterol components of the lipoidal antigen therebyforming an immobilized anchor antibody-lipoidal antigen complex.
 2. Theimmunoassay device of claim 1, wherein the substrate is a microporousmembrane.
 3. The immunoassay device of claim 1, wherein the anchorantibody does not specifically bind an exogenous epitope in one or moreof the cardiolipin, lecithin or cholesterol components of the lipoidalantigen.
 4. The immunoassay device of claim 1, wherein the lipoidalantigen comprises a micelle and not a liposome.
 5. The immunoassaydevice of claim 1, wherein the anchor antibody is specifically bound tothe cardiolipin component of the lipoidal antigen.
 6. The immunoassaydevice of claim 1 for determining at least one of the presence or amountof an anti-lipoidal antibody in a fluid sample, further comprising asample application area and a flow path from the sample application areato the anti-lipoidal antibody capture area; wherein the at least one ofthe presence or amount of an anti-lipoidal antibody in a fluid samplecan be detected by formation of a complex between an anti-lipoidalantibody in a fluid sample and the immobilized lipoidal antigen-anchorantibody complex.
 7. The immunoassay device of claim 1, furthercomprising a treponemal capture area comprising: (a) an immobilizedtreponemal antigen capable of being specifically bound by an anti-T.pallidum antibody, or (b) an immobilized anti-T. pallidum antibody thatspecifically binds a mobile treponemal antigen.
 8. The immunoassaydevice of claim 1, wherein the substrate comprises nitrocellulose,nylon, polyvinylidene fluoride (PVDF), polyethersulfone, polycarbonate,polyester, cellulose acetate, mixed cellulose esters, or combinationsthereof.
 9. The immunoassay device of claim 1, wherein the anchorantibody is a Fab fragment specific for cardiolipin.
 10. The immunoassaydevice of claim 1, wherein the anchor antibody is a plurality of Fabfragments produced from an immunoglobulin fraction from a subjectinfected with T. pallidum.
 11. The immunoassay device of claim 1,wherein the lipoidal antigen is a USR antigen, a VDRL antigen, or asynthetic VDRL antigen.
 12. The immunoassay device of claim 1, whereinthe anti-lipoidal antibody capture area comprises one or more lines. 13.The immunoassay device of claim 12, wherein the one or more lines have awidth from about 8 mm to about 15 mm.
 14. The immunoassay device ofclaim 1, wherein the device is a flow-through device.
 15. Theimmunoassay device of claim 14, further comprising an absorbent pad,which is in contact with the substrate.
 16. The immunoassay device ofclaim 14, wherein the substrate has a pore size from about 0.2 μm toabout 8 μm.
 17. The immunoassay device of claim 1, wherein the device isa lateral flow device.
 18. The immunoassay device of claim 17, whereinthe substrate has a pore size up to about 12 μm.
 19. The immunoassaydevice of claim 17, wherein the lateral flow device further comprises aconjugate pad located in the flow path, wherein the conjugate padcomprises a mobile or mobilizable detector reagent specific for theanti-lipoidal antibody.
 20. The immunoassay device of claim 19, whereinthe detector reagent comprises gold-conjugated Protein A,gold-conjugated Fc-specific Protein G, or gold-conjugated anti-humanantibody (Fc portion).
 21. The immunoassay device of claim 19, whereinthe conjugate pad further comprises a mobile or mobilizable detectorreagent specific for the anti-T. pallidum antibody or the mobiletreponemal antigen.
 22. The immunoassay device of claim 21, wherein thedetector reagent specific for the anti-T. pallidum antibody comprisesgold-conjugated Protein A, gold-conjugated Fc-specific Protein G, orgold-conjugated anti-human antibody (Fc portion), or the detectorreagent for the mobile treponemal antigen comprises gold-labeledanti-treponemal antigen antibody.
 23. The immunoassay device of claim 1,wherein the lipoidal antigen-anchor antibody complex is immobilized onthe substrate by a method comprising: contacting the lipoidal antigenwith one or more anchor antibodies specific for at least one ofcardiolipin, lecithin, or cholesterol to form the lipoidalantigen-anchor antibody complex; and applying the lipoidalantigen-anchor antibody complex to the substrate.
 24. The immunoassaydevice of claim 1, wherein the lipoidal antigen-anchor antibody complexis immobilized on the substrate by a method, comprising: immobilizing ananchor antibody specific for at least one of cardiolipin, lecithin, orcholesterol on the substrate; blocking non-specific binding sites on thesubstrate; contacting the immobilized anchor antibody with a lipoidalantigen to form a lipoidal antigen-anchor antibody complex; and washingthe substrate to remove any lipoidal antigen not specifically bound bythe anchor antibody.
 25. A method for detecting anti-lipoidal antibodiesin a subject, comprising: applying a biological sample from a subject tothe immunoassay device of claim 1; and detecting the formation of acomplex between an anti-lipoidal antibody present in the biologicalsample and the immobilized lipoidal antigen-anchor antibody complex,wherein detection of the formation of the complex detects theanti-lipoidal antibody in the subject.
 26. The method of claim 25,wherein detection of the anti-lipoidal antibody is used to diagnosesyphilis in the subject.
 27. The method of claim 25, wherein thebiological sample is blood, serum, skin ulcer exudate, urine, saliva,sputum, or cerebrospinal fluid.
 28. The method of claim 25, furthercomprising applying to the immunoassay device a detector reagentspecific for the anti-lipoidal antibody.
 29. The method of claim 25,further comprising adding a detector reagent specific for theanti-lipoidal antibody to the biological sample prior to or concurrentwith applying the sample to the immunoassay device.
 30. The method ofclaim 27 wherein the detector reagent is labeled Protein A, Fc-specificProtein G, or anti-human antibody.
 31. The method of claim 30, whereinthe label is an enzyme, colloidal gold particles, colored latexparticles, a chemiluminescent agent, or a fluorescent agent.
 32. Amethod for diagnosing syphilis in a subject, comprising: applying abiological sample to the device of claim 7; detecting in theanti-lipoidal antibody capture area the formation of a first complexbetween an anti-lipoidal antibody present in the biological sample andthe immobilized lipoidal antigen-anchor antibody complex; and detectingin the treponemal capture area the formation of a second complexbetween: (a) an anti-T. pallidum antibody present in the biologicalsample and the immobilized treponemal antigen, or (b) a treponemalantigen present in the biological sample and the immobilized anti-T.pallidum antibody, wherein detection of the formation of the firstcomplex and the second complex is used to diagnose syphilis in thesubject.
 33. The method of claim 32, wherein the biological sample is ahuman blood sample.
 34. The method of claim 33, wherein the human bloodsample is whole blood or serum.
 35. An immunoassay device fordetermining at least one of presence or amount of an anti-lipoidalantibody in a fluid sample comprising: a microporous membrane; a sampleapplication area; an anti-lipoidal antibody capture area comprising alipoidal antigen-anchor antibody complex, which complex comprises ananchor antibody and a lipoidal antigen specifically bound by the anchorantibody, and which complex is immobilized on the membrane by the anchorantibody; wherein the lipoidal antigen comprises cardiolipin, lecithinand cholesterol; and a flow path from the sample application area to theanti-lipoidal antibody capture area; wherein the presence and/or amountof an anti-lipoidal antibody in a fluid sample applied to the sampleapplication area can be detected by formation of a complex between theanti-lipoidal antibody and the immobilized lipoidal antigen-anchorantibody complex.
 36. A method for immobilizing immunoreactivecardiolipin on a substrate, the method comprising: contacting a lipoidalantigen, comprising immunoreactive cardiolipin, with one or moreantibodies specific for at least one component of the lipoidal antigento form a lipoidal antigen-antibody complex; and applying the lipoidalantigen-antibody complex to a substrate, wherein applying the lipoidalantigen-antibody complex to the substrate immobilizes the immunoreactivecardiolipin on the substrate.
 37. The method of claim 36, wherein thelipoidal antigen is a USR antigen, a VDRL antigen, or a synthetic VDRLantigen.
 38. The method of claim 36, wherein the antibody is an antibodyfragment that will not substantially react with Protein A, Fc-specificProtein G, or anti-human antibody (Fc portion).
 39. The method of claim38, wherein the antibody is a Fab fragment.
 40. The method of claim 36,wherein the antibody is isolated from serum of a T. pallidum-infected orT. pallidum-inoculated subject.
 41. The method of claim 36, wherein thesubstrate comprises nitrocellulose.
 42. The method of claim 36,comprising: contacting a lipoidal antigen, comprising immunoreactivecardiolipin, lecithin, and cholesterol, with an antibody fragmentspecific for cardiolipin, lecithin, or cholesterol to form a lipoidalantigen-antibody complex; wherein the antibody fragment does notsubstantially react with Protein A, Fc-specific Protein G, or anti-humanantibody (Fc portion); and applying the lipoidal antigen-antibodycomplex to a substrate, which immobilizes the immunoreactive cardiolipinon the substrate.
 43. The method of claim 42, comprising contacting alipoidal antigen, comprising immunoreactive cardiolipin, with a Fabfragment specific for cardiolipin, lecithin, or cholesterol to form alipoidal antigen-Fab complex; wherein the lipoidal antigen is a USRantigen, a VDRL antigen, or a synthetic VDRL antigen; and applying thelipoidal antigen-Fab complex to nitrocellulose, which immobilizes theimmunoreactive cardiolipin on the nitrocellulose.
 44. A method forimmobilizing immunoreactive cardiolipin on a substrate, the methodcomprising: immobilizing one or more antibodies specific for at leastone of cardiolipin, lecithin or cholesterol on a substrate; blockingnon-specific binding sites on the substrate; applying a lipoidalantigen, comprising at least one immunoreactive cardiolipin, lecithin orcholesterol, to the substrate to form lipoidal antigen-immobilizedantibody complexes; and washing the substrate to remove any lipoidalantigen that is not specifically bound by the one or more immobilizedantibodies.